Early apoptotic cells for use in treating sepsis

ABSTRACT

Compositions disclosed herein, and methods of use thereof included those for treating or preventing sepsis in a subject in need, including methods of extending of the survival of a subject suffering from sepsis, and reduction of organ dysfunction or failure due to sepsis. Methods of treating or preventing sepsis in a subject in need includes administering compositions comprising early apoptotic cells or early apoptotic cell supernatants. Compositions and methods of use thereof may reduce the negative proinflammatory effect accompanying sepsis. Further, anti-inflammatory cytokine release may be reduced. In certain instances, compositions may include additional agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-part application of U.S. patentapplication Ser. No. 16/672,547 filed Nov. 4, 2019, which is aContinuation-in-part application of U.S. patent application Ser. No.16/594,463 filed Oct. 7, 2019, which is a Continuation-in-partapplication of U.S. patent application Ser. No. 16/194,417, filed Nov.19, 2018, which is a Continuation-in-part application of U.S. patentapplication Ser. No. 15/685,086, filed Aug. 24, 2017, which filed as aContinuation-in-part application of U.S. patent application Ser. No.15/551,284 filed Aug. 16, 2017, which filed as a National PhaseApplication of PCT International Application Number PCT/IL2016/050194,International filing date Feb. 18, 2016, which claims the benefit ofU.S. Provisional Application Ser. No. 62/117,752 filed Feb. 18, 2015,U.S. Provisional Application Ser. No. 62/127,218 filed Mar. 2, 2015,U.S. Provisional Application Ser. No. 62/148,227 filed Apr. 16, 2015,and U.S. Provisional Application Ser. No. 62/159,365 filed May 11, 2015.The U.S. application Ser. No. 15/685,086, is also a Continuation-in-partof PCT International Application Number PCT/IL2017/050196, Internationalfiling date Feb. 15, 2017, which claims the benefit of U.S. ProvisionalApplication Ser. No. 62/296,622 filed Feb. 16, 2016 and U.S. ProvisionalApplication Ser. No. 62/370,741 filed Aug. 4, 2016. The U.S. applicationSer. No. 15/685,086, is also a Continuation-in-part application of PCTInternational Application Number PCT/IL2016/050430, International filingdate Apr. 21, 2016, which claims the benefit of U.S. ProvisionalApplication Ser. No. 62/150,305 filed Apr. 21, 2015. The U.S.application Ser. No. 15/685,086, also claims the benefit of U.S.Provisional Application Ser. No. 62/516,714, filed Jun. 8, 2017. All ofthese applications are hereby incorporated by reference in theirentirety herein.

FIELD OF INTEREST

Disclosed herein are compositions and methods thereof for inhibiting orreducing the incidence of cytokine release syndrome (CRS) or a cytokinestorm in a subject undergoing CAR T-cell cancer therapy. Further,disclosed herein are compositions and methods thereof for decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or a cytokine storm. Further, compositions disclosedherein may be used for treating, preventing, inhibiting the growth of,or reducing the incidence of, a cancer or a tumor in a subject.Compositions may be used for increasing survival of a subject sufferingfrom a cancer or a tumor. Compositions used may be administered alone orin combination with other chemotherapies. Methods disclosed hereininclude those comprising administration of a composition comprisingapoptotic cells or an apoptotic cell supernatant alone or in combinationwith a CAR T-cell therapy. Methods disclosed herein include those totreat sepsis.

BACKGROUND

While standard treatments for cancer are surgery, chemotherapy, andradiation therapy, improved methods, such as targeted immunologicaltherapies, are currently being developed and tested. One promisingtechnique uses adoptive cell transfer (ACT), in which immune cells aremodified to recognize and attack their tumors. One example of ACT iswhen a patient's own cytotoxic T-cells, or a donor's, are engineered toexpress a chimeric antigen receptor (CAR T-cells) targeted to a tumorspecific antigen expressed on the surface of the tumor cells. These CART-cells are then cytotoxic only to cells expressing the tumor specificantigen. Clinical trials have shown that CAR T-cell therapy has greatpotential in controlling advanced acute lymphoblastic leukemia (ALL) andlymphoma, among others.

However, some patients given CAR T-cell therapy and other immunetherapies experience a dangerous and sometimes life-threatening sideeffect called cytokine release syndrome (CRS), in which the infused,activated T-cells produce a systemic inflammatory response in whichthere is a rapid and massive release of cytokines into the bloodstream,leading to dangerously low blood pressure, high fever and shivering.

In severe cases of CRS, patients experience a cytokine storm (a.k.a.cytokine cascade or hypercytokinemia), in which there is a positivefeedback loop between cytokines and white blood cells with highlyelevated levels of cytokines. This can lead to potentiallylife-threatening complications including cardiac dysfunction, adultrespiratory distress syndrome, neurologic toxicity, renal and/or hepaticfailure, pulmonary edema and disseminated intravascular coagulation.

For example, six patients in a recent phase I trial who wereadministered the monoclonal antibody TGN1412, which binds to the CD28receptor on T-cells, exhibited severe cases of cytokine storm andmulti-organ failure. This happened despite the fact that the TGN1412dose was 500-times lower than that found to be safe in animals (St.Clair E W: The calm after the cytokine storm: Lessons from the TGN1412trial. J Clin Invest 118: 1344-1347, 2008).

To date, corticosteroids, biological therapies such as anti-IL6therapies and anti-inflammatory drugs are being evaluated to controlcytokine release syndrome in patients administered CAR T-cell therapy.However, steroids may affect CAR T-cells' activity and/or proliferationand put the patients in danger of sepsis and opportunistic infections.Anti-inflammatory drugs may not be effective in controlling cytokinerelease syndromes or cytokine storms, because the cytokine stormincludes a very large number of cytokines while there is limited abilityto infuse patients with anti-inflammatory drugs. Novel strategies areneeded to control cytokine release syndromes, and especially cytokinestorms, in order to realize the potential of CAR T-cell therapy.

Cytokine storms are also a problem after other infectious andnon-infectious stimuli. In a cytokine storm, numerous proinflammatorycytokines, such as interleukin-1 (IL-1), IL-6, g-interferon (g-IFN), andtumor necrosis factor-α (TNFα), are released, resulting in hypotension,hemorrhage, and, ultimately, multiorgan failure. The relatively highdeath rate in young people, with presumably healthy immune systems, inthe 1918 H1N1 influenza pandemic and the more recent bird flu H5N1infection are attributed to cytokine storms. This syndrome has been alsoknown to occur in advanced or terminal cases of severe acute respiratorysyndrome (SARS), Epstein-Barr virus-associated hemophagocyticlymphohistiocytosis, sepsis, gram-negative sepsis, malaria and numerousother infectious diseases, including Ebola infection.

Cytokine storm may also stem from non-infectious causes, such as acutepancreatitis, severe burns or trauma, or acute respiratory distresssyndrome. Novel strategies are therefore needed to control cytokinerelease syndrome, and especially cytokine storms.

Cancer is an abnormal state in which uncontrolled proliferation of oneor more cell populations interferes with normal biological functioning.The proliferative changes are usually accompanied by other changes incellular properties, including reversion to a less differentiated, moredevelopmentally primitive state. The in vitro correlate of cancer iscalled cellular transformation. Transformed cells generally displayseveral or all of the following properties: spherical morphology,expression of fetal antigens, growth-factor independence, lack ofcontact inhibition, anchorage-independence, and growth to high density.

The primary cause of lethality of malignant diseases such as lung andskin cancer arise from metastatic spread. In many cases, it is notpossible to prevent the onset of metastatic disease since cancers areoften metastatic by the time of diagnosis, and even in cases wherecancers are diagnosed prior to this stage, complete surgical removal ordestruction of primary lesion tissues which are capable of eventuallygenerating metastases may not be feasible. Metastatic disease may beimpossible to diagnose at early stages due to the small size ofmetastatic lesions, and/or the absence of reliable markers in primarylesions upon which to reliably predict their existence. Such lesions maybe difficult or impossible to treat via ablative methods due to theirbeing inaccessible, disseminated, and/or poorly localized.Chemotherapy/radiotherapy, the current methods of choice for treatmentof certain metastatic malignancies are often ineffective or suboptimal,and have the significant disadvantage of being associated withparticularly harmful and/or potentially lethal side-effects.

Immunotherapeutic cancer treatment methods, such as those involvingantigen presenting cell (APC) vaccinations, have the potential to beoptimally effective for treatment of inaccessible, disseminated,microscopic, recurrent and/or poorly localized cancer lesions. Onepromising immunotherapy avenue involves the use of professional APCs,such as dendritic cells (DCs), to elicit systemic anti-cancer immunity.

Dendritic cells are antigen-producing and presenting cells of themammalian immune system that process antigen material and present it onthe cell surface to the T-cells of the immune system and are therebycapable of sensitizing T-cells to both new and recall antigens. DCs arethe most potent antigen-producing cells, acting as messengers betweenthe innate and the adaptive immune systems. DC cells may be used, toprime specific antitumor immunity through the generation of effectorcells that attack and lyse tumors.

Sepsis is the body's overwhelming and life-threatening response toinfection that can lead to tissue damage, organ failure and death. Inother words, it's a body's overactive and toxic response to aninfection.

The immune system usually works to fight any germs (bacteria, viruses,fungi or parasites) to prevent infection. If an infection does occur,the immune system will try to fight it, although it may need help frommedication such as antibiotics, antivirals, antifungals andantiparasitics. However, for reasons researchers do not understand, theimmune system sometimes stops fighting the “invaders,” and begins toturn on itself. This is the start of sepsis.

People who are at higher risk of developing sepsis are generally peoplewho are at higher risk of contracting an infection. These could includethe very young, the very old, those with chronic illnesses and thosewith a weakened or impaired immune system. Patients are diagnosed withsepsis when they develop a set of signs and symptoms related to sepsis.Sepsis is not diagnosed based on an infection itself. If a person hasmore than one of the symptoms of sepsis, especially if there are signsof an infection or if someone falls into one of the higher risk groups,the physician will likely suspect sepsis.

Sepsis, which has been identified by the World Health Organization (WHO)as a global health priority, has no proven pharmacologic treatment otherthan appropriate antibiotic agents, fluids, vasopressors as needed, andpossibly corticosteroids (Venkatesh, B., Finfer, S., Cohen, J.,Rajbhandari, D., Arabi, Y., Bellomo, R., Billot, L., Correa, M., Glass,P., Harward, M., et al. (2018). Adjunctive Glucocorticoid Therapy inPatients with Septic Shock. N. Engl. J. Med. 378, 797-808). Reporteddeath rates among hospitalized patients range between 30% and 45%(Finfer, S., Bellomo, R., Lipman, J., French, C., Dobb, G., and Myburgh,J. (2004). Adult-population incidence of severe sepsis in Australian andNew Zealand intensive care units. Intensive Care Med. 30, 589-596;Fleischmann, C., Scherag, A., Adhikari, N. K. J., Hartog, C. S.,Tsaganos, T., Schlattmann, P., Angus, D. C., Reinhart, K., andInternational Forum of Acute Care Trialists (2016). Assessment of GlobalIncidence and Mortality of Hospital-treated Sepsis. Current Estimatesand Limitations. Am. J. Respir. Crit. Care Med. 193, 259-272; Liu, V.,Escobar, G. J., Greene, J. D., Soule, J., Whippy, A., Angus, D. C., andIwashyna, T. J. (2014). Hospital Deaths in Patients With Sepsis From 2Independent Cohorts. JAMA 312, 90; Machado, F. R., Cavalcanti, A. B.,Bozza, F. A., Ferreira, E. M., Angotti Carrara, F. S., Sousa, J. L.,Caixeta, N., Salomao, R., Angus, D. C., Pontes Azevedo, L. C., et al.(2017). The epidemiology of sepsis in Brazilian intensive care units(the Sepsis PREvalence Assessment Database, SPREAD): an observationalstudy. Lancet Infect. Dis. 17, 1180-1189; Reinhart, K., Daniels, R.,Kissoon, N., Machado, F. R., Schachter, R. D., and Finfer, S. (2017).Recognizing Sepsis as a Global Health Priority—A WHO Resolution. N.Engl. J. Med. 377, 414-417; Rhee, C., Dantes, R., Epstein, L., Murphy,D. J., Seymour, C. W., Iwashyna, T. J., Kadri, S. S., Angus, D. C.,Danner, R. L., Fiore, A. E., et al. (2017). Incidence and Trends ofSepsis in US Hospitals Using Clinical vs Claims Data, 2009-2014. JAMA318, 1241).

Sepsis is generally initiated by simultaneous recognition of eitherpathogen-associated molecular patterns (PAMPs) or damage-associatedmolecular patterns (DAMPs) by components of the innate immune system,including complement proteins, Toll-like receptors, NOD-like receptors,RIG-like receptors, mannose-binding lectin, and scavenger receptors(HOTShkiss, R. S., Moldawer, L. L., Opal, S. M., Reinhart, K., Turnbull,I. R., and Vincent, J.-L. (2016). Sepsis and septic shock. Nat. Rev.Dis. Prim. 2, 16045). Recognition induces a complex intracellularsignaling system with redundant and complementary activities, andactivation of these multiple signaling pathways ultimately leads to theexpression of several common classes of genes that are involved ininflammation, adaptive immunity, and cellular metabolism (Tang, D.,Kang, R., Coyne, C. B., Zeh, H. J., and Lotze, M. T. (2012). PAMPs andDAMPs: signal 0s that spur autophagy and immunity. Immunol. Rev. 249,158-175).

Sepsis elicits dysregulated immune responses manifested by acytokine/chemokine elevation (also known as ‘cytokine storm’) thatcorrelates well with disease severity and poor prognosis (Chaudhry, H.,Zhou, J., Zhong, Y., Ali, M. M., McGuire, F., Nagarkatti, P. S., andNagarkatti, M. (2015). Role of cytokines as a double-edged sword insepsis. In Vivo 27, 669-684; Matsumoto, H., Ogura, H., Shimizu, K.,Ikeda, M., Hirose, T., Matsuura, H., Kang, S., Takahashi, K., Tanaka,T., and Shimazu, T. (2018). The clinical importance of a cytokinenetwork in the acute phase of sepsis. Sci. Rep. 8, 1-4). Thisexaggerated immune response deleteriously affects physiologicalhomeostasis of vital organs, including the kidney, liver, lungs, andheart, and often evolves into multi-organ failure, also termed MultipleOrgan Dysfunction Syndrome (MODS) (Marshall, J. C., Cook, D. J.,Christou, N. V, Bernard, G. R., Sprung, C. L., and Sibbald, W. J.(1995). Multiple organ dysfunction score: a reliable descriptor of acomplex clinical outcome. Crit. Care Med. 23, 1638-16525; Vincent, J.-L.(2006). Organ Dysfunction in Patients with Severe Sepsis. Surg. Infect.(Larchmt). 7, s-69-s-72).

Sepsis progresses to severe sepsis when, in addition to signs of sepsis,the patient experiences indications of organ dysfunction, such asdifficulty breathing (lungs), low or no urine output (kidneys), abnormalliver tests (liver), and changes in mental status (brain). Nearly allpatients with severe sepsis require treatment in an intensive care unit(ICU). Septic shock is the most severe level and is diagnosed when apatient's blood pressure drops to dangerous levels.

The current treatment for sepsis includes: the administration ofantibiotics and, when indicated, surgical or interventional radiologicalapproaches for eliminating or at least controlling the source ofinfection; the administration of intravenous fluids (crystalloidsolutions such as 0.9% sodium chloride solution, or colloid solutionssuch as 5% albumin solution) to restore and maintain adequateintravascular volume; the infusion of titratable vasoconstricting and/orinotropic drugs, such as vasopressin or noradrenaline, as needed, tochange the strength of a heart's contractions; and, when indicated,mechanical ventilation, various forms of renal replacement therapy and,in rare cases, venovenous or venoarterial extracorporeal membraneoxygenation.

Due to the high rates of mortality and morbidity from sepsis, and theassociated economic burden, the need for a novel pharmacological therapyis obvious. Unfortunately, a recent study examining the role ofglucocorticoids in patients with septic shock who were undergoingmechanical ventilation, found that administration of a continuousinfusion of hydrocortisone did not result in lower 90-day mortalitycompared to placebo.

It appears that previous attempts to find a therapy for sepsis faileddue to the parallel course of biological activities that occur within asepsis patient. While the medical team is administering the beststandard of care, mainly antibiotics, a Cytokine Release Syndrome rampsup at the same time. A Cytokine Release Syndrome is difficult to treatwith traditional small molecules or biotech drugs as the conditioninvolves dozens of cytokines that induce multiple biological paths ofhyper immune activity. Such hyper immune activity may result in anattack of immune killer cells (e.g., T-Cells, B-Cells, Natural KillerCells) on healthy organs of the patient, such as heart, brain, lungs,liver, kidney and others. This outcome of this attack may lead to organdamage, multiple organ failure and mortality. If the Cytokine ReleaseSyndrome could be prevented, the medical team would have ample time toeradicate the core source of the sepsis (i.e., an antibiotic-resistantbacteria), and most likely significantly increase the patient's chanceof survival and survival statistics.

Apoptotic cells present one pathway of physiological cell death, mostcommonly occurring via apoptosis, which elicits a series of molecularhomeostatic mechanisms comprising recognition, an immune response and aremoval process. Moreover, apoptotic cells are immunomodulatory cellscapable of directly and indirectly inducing immune tolerance todendritic cells and macrophages. Apoptotic cells have been shown tomodulate dendritic cells and macrophages and to render them tolerogenicand inhibit proinflammatory activies such as secretion ofproinflammatory cytokines and expression of costimulatory molecules.

As many as 3×10⁸ cells undergo apoptosis every hour in the human body.One of the primary “eat me” signals expressed by apoptotic cells isphosphatidylserine (PtdSer) membrane exposure. Apoptotic cellsthemselves are major contributors to the “non-inflammatory” nature ofthe engulfment process, some by secreting thrombospondin-1 (TSP-1) oradenosine monophosphate and possibly other immune modulating “calm-down”signals that interact with macrophages and DCs. Apoptotic cells alsoproduce “find me” and “tolerate me” signals to attract andimmunomodulate macrophages and DCs that express specific receptors forsome of these signals.

The pro-homeostatic nature of apoptotic cell interaction with the immunesystem is illustrated in known apoptotic cell signaling events inmacrophages and DCs that are related to Toll-like receptors (TLRs),NF-κB, inflammasome, lipid-activated nuclear receptors, Tyro3, Axl, andMertk receptors. In addition, induction of signal transducers,activation of transcription 1, and suppression of cytokine signalinglead to immune system silencing and DC tolerance Trahtemberg, U., andMevorach, D. (2017). Apoptotic cells induced signaling for immunehomeostasis in macrophages and dendritic cells. Front. Immunol. 8).

As summarized recently (Trahtemberg, U., and D. Mevorach. 2017.Apoptotic cells induced signaling for immune homeostasis in macrophagesand dendritic cells. Front Immunol 8:1356), apoptotic cells may have abeneficial effect on aberrant immune response, with downregulation ofboth anti- and pro-inflammatory cytokines derived from PAMPs and DAMPs,in both animal and in vitro models.

There remains an unmet need for compositions and methods for treating,preventing, inhibiting the growth of, or reducing the incidence of, acancer or a tumor in a subject. The apoptotic cell preparations,compositions and uses thereof, described herein below, address this needby providing a population of early apoptotic cells that may be used totreat, prevent, inhibit the growth or, or reduce the incidence of acancer or tumor in a subject. Further, the methods of use describedherein address the need to increasing survival of a subject sufferingfrom a cancer and tumor, including increasing remission of the cancer ortumor.

Further, there remains an unmet need for compositions and methods oftreatment of sepsis, including for the prevention of organ failure andmortality in patients with sepsis.

The methods of use described herein addresses the need to increasingsurvival of a subject suffering from sepsis, and provides an unexpectedsolution for treating sepsis, including preventing organ failure.

SUMMARY

In one aspect disclosed herein is a method of treating, preventing,inhibiting, reducing the incidence of, ameliorating, or alleviatingsepsis, or any combination thereof, in a subject in need, comprising thestep of administering a composition comprising an early apoptotic cellpopulation to said subject, wherein said administering treats, prevents,inhibits, reduces the incidence of, ameliorates, or alleviates sepsis insaid subject.

In a related aspect, the sepsis comprises mild or severe sepsis. In someembodiments, the source of sepsis comprises pneumonia, an endovascularmethicillin-resistant Staphylococcus aureus (MRSA) infection, or aurinary tract infection (UTI).

In another related aspect, the method results in increased survival ofsaid subject. In another related aspect, the incidence of organ failureor organ dysfunction, or organ damage, or a combination thereof, in asubject treated by the method, is reduced. In a further related aspect,the organ failure comprises acute multiple organ failure.

In a related aspect, the early apoptotic cell population comprises

(a) a mononuclear enriched cell population; or (b) an apoptoticpopulation stable for greater than 24 hours; or (c) a mononuclearapoptotic cell population comprising a decreased of non-quiescentnon-apoptotic cells, a suppressed cellular activation of any livingnon-apoptotic cells, or a reduced proliferation of any livingnon-apoptotic cells, or any combination thereof; any combinationthereof. In a related aspect, the early apoptotic cell populationcomprises a pooled population of early apoptotic cells.

In a related aspect, the subject in need is a human subject.

In a related aspect, the administering comprises a single infusion ofsaid early apoptotic cell population. In a further related aspect, theadministering comprises multiple infusions of said apoptotic cellpopulation. In an additional related aspect, the administering comprisesintra venal administration.

In a related aspect, the method further comprises administering anadditional therapy. In a further related aspect, the additional therapyis administered prior to, concurrent with, or following administrationof said early apoptotic cells.

In a related aspect, method comprises a first-line therapy. In anotherrelated aspect, the method comprises an adjuvant therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The subject matter disclosed herein is particularly pointed out anddistinctly claimed in the concluding portion of the specification. Thecompositions and methods disclosed herein, however, both as toorganization and method of operation, together with objects, features,and advantages thereof, may best be understood by reference to thefollowing detailed description when read with the accompanying drawings.

FIGS. 1A-1B. Schematic showing standard CAR T-cell therapy (FIG. 1A) andembodiments of a method of safe and efficacious CAR T-cell cancertherapy in a patient using patients' own cells (autologous) (FIG. 1B) toproduce apoptotic cells or an apoptotic cell supernatant.

FIG. 2 . Schematic showing embodiments of a method of safe andefficacious CAR T-cell cancer therapy in a patient, using donor cells toproduce apoptotic cells or an apoptotic supernatant.

FIG. 3 . Flow chart presenting the steps during one embodiment of amanufacturing process of an early apoptotic cell population, whereinanti-coagulants were included in the process.

FIGS. 4A-4J. Apoptotic cells prevent cytokine storm in in vitro model ofcytokine storm induced in LPS-Sterile model of macrophage activationsyndrome in a cancer environment. FIG. 4A shows the reduction of LPSinduced IL-10 levels in the macrophage activation syndrome model in thepresence of cancer following administration of Apocells at amacrophage/monocyte:Apocell ratio of 1:8 and 1:16, at two time periods(6 hours and 24 hours). FIG. 4B shows the reduction of LPS induced IL-6levels in the macrophage activation syndrome model followingadministration of Apocells in the presence of cancer and CAR-19, at amacrophage/monocyte:Apocell ratio of 1:8 and 1:16, at two time periods(6 hours and 24 hours). FIG. 4C shows the reduction of LPS inducedMIP-1α levels in the macrophage activation syndrome model in thepresence of cancer and CAR-19, following administration of Apocells at amacrophage/monocyte:Apocell ratio of 1:8 and 1:16, at two time periods(6 hours and 24 hours). FIG. 4D shows the reduction of LPS induced IL-8levels in the macrophage activation syndrome model in the presence ofcancer and CAR-19, following administration of Apocells at amacrophage/monocyte:Apocell ratio of 1:8 and 1:16, at two time periods(6 hours and 24 hours). FIG. 4E shows the reduction of LPS induced TNF-αlevels in the macrophage activation syndrome model in the presence ofcancer and CAR-19, following administration of Apocells at amacrophage/monocyte:Apocell ratio of 1:8 and 1:16, at TWO time periods(6 hours and 24 hours). FIG. 4F shows the reduction of LPS inducedMIP-10 levels in the macrophage activation syndrome model in thepresence of cancer and CAR-19, following administration of Apocells at amacrophage/monocyte:Apocell ratio of 1:4, 1:8, 1:16, 1:32, and 1:64 at24 hours. FIG. 4G shows the reduction of LPS induced MCP-1 levels in themacrophage activation syndrome model in the presence of cancer andCAR-19, following administration of Apocells at amacrophage/monocyte:Apocell ratio of 1:4, 1:8, 1:16, 1:32, and 1:64 at24 hours. FIG. 4H shows the reduction of LPS induced IL-9 levels in themacrophage activation syndrome model in the presence of cancer andCAR-19, following administration of Apocells at amacrophage/monocyte:Apocell ratio of 1:8 and 1:16, at two time periods(6 hours and 24 hours). FIG. 4I shows the increase of LPS induced IL-2Rlevels in the macrophage activation syndrome model in the presence ofcancer and CAR-19, following administration of Apocells at amacrophage/monocyte:Apocell ratio of 1:4, 1:8, 1:16, 1:32, and 1:64 at24 hours. FIG. 4J shows that apoptotic cells do not down regulate IL-2release from cells. Apoptotic cells were incubated withmacrophages/monocytes in the presence of cancer and CAR-19, over a 24hour time period with increasing doses of apoptotic cells (n=3). Emptybar (outline only)—2.5×10⁶ apoptotic cells per well; Black—5×10⁶apoptotic cells per well; Grey—10×10⁶ apoptotic cells per well.

FIG. 5 . Verification of Transduction of T-cells showing the flowcytometry results of anti-CD124 analysis of transduced T4⁺ CAR-T cells.

FIG. 6 . T4⁺ CAR T-Cells reduced proliferation of SKOV3-luc ovarianadenocarcinoma cells. The results of the cytotoxicity assay, wherein amonolayer of SKOV3-luc cells were cultured either by non-transduced Tcells or by T4+ CAR-T cells, are presented in a bar graph.

FIG. 7 . Apoptotic Cells do not abrogate T4⁺ CAR-T cells anti-tumoractivity. Results are based on a cytotoxicity assay, wherein a monolayerof SKOV3-luc cells were cultured either with non-transduced T cells orwith T4⁺CAR-T cells in the presence of a vehicle (Hartmann solution), orapoptotic cells (Apocell), or a supernatant of apoptotic cells (ApoSup),or supernatant of co-culture of apoptotic cells andmonocytes/macrophages (ApoMon Sup).

FIG. 8 . Il-6, secreted at high levels during cytotoxicity, isdown-regulated by apoptotic cells. The results shown here demonstratethe effect of co-culture of SKOV3-luc and human monocytes/macrophageswere exposed to apoptotic cells (ApoCell), or ApoCell supernatant(ApoSup), or apoptotic cells and monocyte/macrophage co-culture (ApoMonSup).

FIG. 9 . Effect of Apoptotic Cells or Apoptotic Cell Supernatant or aco-culture of Apoptotic cells and Monocytes following LPS exposureduring CAR-T cell therapy. Extremely high secretion of IL-6 wasdocumented when lipopolysaccharides (LPS) were added to the cytotoxicassay. Results show that exposure to Apoptotic cells (Apocell), orsupernatant of apoptotic cells (ApoSup) or supernatant of co-culture ofapoptotic cells and monocytes/macrophages (ApoMon Sup), down regulatedIL-6, wherein IL-6 was reduced to acceptable levels.

FIG. 10 . Effect of Apoptotic Cells or Apoptotic Cell Supernatant or aco-culture of Apoptotic cells and Monocytes following LPS exposureduring CAR T-cell treatment mimicking CAR T-cell clinical therapy.Extremely high secretion of IL-6 was documented when lipopolysaccharidesLPS) were added to the cytotoxic assay. Results show that exposure toApoptotic cells (Apocell), or supernatant of apoptotic cells (ApoSup) orsupernatant of co-culture of apoptotic cells and monocytes/macrophages(ApoMon Sup), down regulated IL-6, wherein IL-6 was reduced toacceptable levels.

FIGS. 11A-11B. Weight and Tumor Size in Mice at time of Culling. FIG.11A shows Weight change over the experimental time period. Blue-controlno 4.5×10⁶ SKOV3-luc cells administrated. Red—0.5×10⁶ SKOV3-luc cells.Green-1.0×10⁶ SKOV3-luc cells. Purple-4.5×10⁶ SKOV3-luc cells FIG. 11Bpresents a representative SKOV3-luc tumor for a mouse receiving 4.5×10⁶SKOV3-luc cells, 39 days after injection.

FIG. 12 . SKOV3-luc Tumor Growth. Mice bearing SKOV3-luc tumors imagedby Bioluminescent imaging (BLI) are presented showing the differencesbetween control (PBS) and inoculation with 0.5×10⁶, 1×10⁶, and 4.5×10⁶SKOV3-luc cells.

FIGS. 13A-13D. SKOV3-luc Tumor Burden. Quantification of bioluminescence(BLI) of SKOV3-luc tumors in vivo (See FIG. 12 ). A 600 photon countcut-off was implemented as instructed by the manufacturer. FIG. 13A,mice inoculated with 0.5×106 SKOV3-luc. FIG. 13B, mice inoculated with1×106 SKOV3-luc. FIG. 13C, mice inoculated with 4.5×106 SKOV3-luc.

FIG. 13D, Average SKOV3-luc tumor growth.

FIG. 14 . Cytotoxic Calibration for Raji Burkett Lymphoma Cells. Rajicells were plated at various cell densities, with cell lysis occurringimmediately prior to centrifugation. The results show Raji cell number(x-axis) vs. at absorbance at 492 nm (y-axis). All cell numbersexhibited significant readings relative to the unlysed counterpart.

FIG. 15 . Addition of early apoptotic cells does not affect CAR T-cellanti-tumor activity. E/T ratio shows the CD19+CAR T-cell to HeLa cellratio. Survival is of CD19+ Tumor cells. Filled circle CD19+ Hela; Emptytriangle CD19+ Hela+ Naïve T cells; Filled triangle CD19+ Hela+ CART-CD19; Empty circle CD19+ Hela+ CAR T-CD19+ApoCells.

FIG. 16 . Cytokine Analysis (GM-CSF) in Raji Burkett Lymphoma Cells inthe Presence and Absence of Apoptotic cells. The bar graph presents theconcentration measurements of cytokine GM-CSF (pg/ml) found in culturesupernatants of Raji cells incubated in the presence of monocytes andLPS, followed by addition of Naïve T-cells (Raji+Naïve T), CD19+ CART-cells (Raji+CAR T), CD19+ CAR T-cells and apoptotic cells (ApoCell) ata ratio of 1:8 CAR T-cells:ApoCells (Raji+CAR T+ApoCell 1:8), CD19+ CART-cells and apoptotic cells (ApoCell) at a ratio of 1:32 CART-cells:ApoCells (Raji+CAR T+ApoCell 1:32), and CD19+ CAR T-cells andapoptotic cells (ApoCell) at a ratio of 1:64 CAR T-cells:ApoCells(Raji+CAR T+ApoCell 1:64).

FIG. 17 . Cytokine Analysis (TNF-alpha) in Raji Burkett Lymphoma Cellsin the Presence and Absence of Apoptotic cells. The bar graph presentsthe concentration measurements of cytokine TNF-alpha (TNF-a) (pg/ml)found in culture supernatants of Raji cells incubated in the presence ofmonocytes and LPS, followed by addition of Naïve T-cells (Raji+Naïve T),CD19+ CAR T-cells (Raji+CAR T), CD19+ CAR T-cells and apoptotic cells(ApoCell) at a ratio of 1:8 CAR T-cells:ApoCells (Raji+CAR T+ApoCell1:8), CD19+ CAR T-cells and apoptotic cells (ApoCell) at a ratio of 1:32CAR T-cells:ApoCells (Raji+CAR T+ApoCell 1:32), and CD19+ CAR T-cellsand apoptotic cells (ApoCell) at a ratio of 1:64 CAR T-cells:ApoCells(Raji+CAR T+ApoCell 1:64).

FIGS. 18A and 18B. Experimental Scheme. FIG. 18A presents theexperimental scheme to analyze the influence of apoptotic cells on CART-cell therapy. SCID mice were injected on day 1 with Raji cancer cells,followed on day 6 by administration of CAR T-CD19 cells (CAR T-celltherapy) and Apoptotic cells. FIG. 18B shows that CAR T-cell therapy wasnot negatively influenced by co-administration of ApoCells. SurvivalCurve: SCID mice were injected with CD19+ Raji cells with or withoutaddition of early apoptotic cells.

FIGS. 19A, 19B, and 19C. Increased release of pro-inflammatory cytokinesfrom a tumor, in a solid tumor in vivo model. FIG. 19A shows slightincrease of IL-6 released from a solid tumor present in the peritoneumof BALB/c and SCID mice, wherein the IL-6 release is significantlyincreased in the presence of HeLa CAR-CD-19 CAR T-cells. Similarly, FIG.19B shows a slight increase of IP-10 released from a solid tumor presentin the peritoneum of BALB/c and SCID mice, wherein the IP-10 release issignificantly increased in the presence of HeLa CAR-CD-19 CAR T-cells,and FIG. 19C shows that surprisingly even TNF-α release is increased byin the presence of HeLa CAR-CD-19 CAR T-cells.

FIGS. 20A and 20B. Testing the efficacy of CD19-CAR-T cells in an IPmodel of HeLaCD19 (Leukemia), in the presence or absence of ApoCell.HeLa-CD19—Blue; HeLaCD19+Mock—Green; HeLaCD19+CAR-T—Purple; andHeLaCD19+CAR-T+ApoCell—orange. FIG. 20A was with 0.5×10⁶ CAR-T positivecells. FIG. 20B was with 2.2×10⁶ CAR-T positive cells.

FIG. 21 . Survival curves for in vivo diffuse tumor SCID mouse model.The curves show that administration of early apoptotic cells (APO; broaddashed lines ----) extended survival compared with mice not administeredapoptotic cells (NO APO; dotted line ⋅⋅⋅⋅), wherein control SCID miceshowed 100% survival (solid line ______).

FIGS. 22A-22D. Apoptotic cell infusions increased the lifespan ofleukemic mice and increased the number of mice attaining completeremission. Cohorts: No leukemia (Control-striped pattern);Leukemia+early apoptotic cells (spotted); Leukemia only (solid grey).n=51 in total (p<0.001) FIG. 22A. Apoptotic cell infusions increased thepercentage of mice surviving through the expected life-span postleukemia induction. FIG. 22B. Apoptotic cell infusions increased thepercentage of mice surviving up to 12% of the expected life-span postleukemia induction. FIG. 22C. Apoptotic cell infusions increased thepercentage of mice surviving up to 30% of the expected life-span postleukemia induction. FIG. 22D. Apoptotic cell infusions increased thepercentage of mice surviving up to 100% of the expected life-span postleukemia induction and attaining complete remission.

FIGS. 23A-23E. Apoptotic cell infusions increased the life-span ofleukemic mice, increased the number of mice attaining completeremission, and enhanced the anti-CD20 monoclonal antibody (mAb)therapeutic effect. Cohorts: Leukemia only (solid grey); Leukemia+earlyapoptotic cells (striped pattern); Leukemia+anti-CD20 mAb (checkered);Leukemia+anti-CD20+early apoptotic cells (spotted). n=28 in total(p<0.002) FIG. 23A. Shows the percent (%) survival through the expectedlifespan of mice following induction of leukemia with Raji cells. FIG.23B. Apoptotic cell infusions increased the percentage of mice survivingup to 24% longer than the expected life-span post leukemia induction.FIG. 23C. Apoptotic cell infusions increased the percentage of micesurviving up to 59% longer than the expected life-span post leukemiainduction and enhanced the anti-CD20 mAb effect on the life-span ofleukemic mice. FIG. 23D. Apoptotic cell infusions increased thepercentage of mice surviving up to 76% longer than the expectedlife-span post leukemia induction and enhanced the anti-CD20 mAb effecton the life-span of leukemic mice. FIG. 23E. Apoptotic cell infusionsincreased the percentage of mice attaining complete remission.

FIG. 24 . Kaplan-Meier survival plot of SCID-Bg mice with Rajileukemia/lymphoma, receiving ApoCell. (RPMI group, n=15; Raji group,n=23; Raji+ApoCell group, n=24) RPMI (control)—Black; Raji only—Orange;Raji+ApoCell—Blue.

FIGS. 25A-25C. Kaplan-Meier survival plots. FIG. 25A presents data froma study wherein female SCID-Bg mice, 7-weeks-old (ENVIGO, Jerusalem,Israel), were injected IV with 0.1×10⁶ Raji cells per mouse (n=10 pergroup, three groups). Mice received three IV doses (days 5, 8, 11) of30×10⁶ ApoCell. (RPMI-light blue; Raji-orange; and Raji+ApoCell—darkblue) FIG. 25B presents data from a study wherein female SCID-Bg mice,7-weeks-old (ENVIGO, Jerusalem, Israel), were injected IV with 0.1×10⁶Raji cells per mouse (n=10 per group, three groups). Mice received threeIV doses (days 5, 8, 11) of 30×10⁶ ApoCell. (RPMI-black; Raji-orange;and Raji+ApoCell—dark blue) FIG. 25C presents data from a study whereinfemale SCID-Bg mice, 8-9-weeks-old (ENVIGO, Jerusalem, Israel), wereinjected IV with 0.1×10⁶ Raji cells per mouse (n=10 per group, 2groups). Mice received three IV doses (days 5, 8, 12) of 30×10⁶ ApoCell.(Raji-orange; and Raji+ApoCell—dark blue)

FIG. 26 . Kaplan-Meier survival plot of SCID-Bg mice with Rajileukemia/lymphoma, receiving RtX and ApoCell. (Raji alone—orange;Raji+ApoCell—blue; Raji+RtX 2 mg—green; Raji+RtX 2 mg+ApoCell—yellow;Raji+RtX 5 mg—purple; Raji+RtX 5 mg+ApoCell—grey.)

FIG. 27 . Kaplan-Meier survival plot of SCID-Bg mice with Rajileukemia/lymphoma, receiving rtx and ApoCell. (Raji alone—orange;Raji+ApoCell—blue; Raji+RtX 2 mg—green; Raji+RtX 2 mg+ApoCell—yellow.)

FIG. 28 . Effect of Pooled ApoCell Preparation. FIG. 28 presents a graphshowing the clear effect (p<0.01) of a single apoptotic cell preparationinjection from multiple individual donors (blue) on survival. The graphpresented is a Kaplan-Meier survival curve in a GvHD mouse model thatwas treated with a single dose irradiated pooled apoptotic cellpreparation from multiple individual donors.

FIG. 29 . Effect of Pooled ApoCell Preparation. FIG. 29 presents a graphshowing the clear effect (p<0.01) of a single apoptotic cell preparationinjection from multiple individual donors (blue) on percentage of weightloss of the 2 compared groups.

FIG. 30 . Comparison of Single Donor versus Pooled ApoCell Preparation.FIG. 30 presents a graph showing comparison between the administrationof a single dose of single-donor and multiple-donor apoptotic cellpreparations+/−irradiation on % survival using a mouse model of inducedGvHD.

FIGS. 31A-31B. Potency Test. FIGS. 31A-31B present the results of apotency test that shows the inhibition of maturation of dendritic cells(DCs) following interaction with apoptotic cells, measured by expressionof HLA-DR. FIG. 31A. HLA DR mean fluorescence of fresh final product A(t0). FIG. 31B. HLA DR mean fluorescence of final product A, following24 h at 2-8° C.

FIGS. 32A-32B. Potency Test. FIGS. 32A-32B present the results of apotency test that shows the inhibition of maturation of dendritic cells(DCs) following interaction with apoptotic cells, measured by expressionof CD86. FIG. 32A. CD86 Mean fluorescence of fresh final product A (t0).FIG. 32B. CD86 Mean fluorescence of final product A, following 24 h at2-8° C.

FIGS. 33A-33E. Results of preclinical analysis of use of early apoptoticcells in the treatment of sepsis. FIG. 33A shows increased survival inmice receiving antibiotic and Allocetra-OTS (early apoptotic cells asdescribed herein). FIG. 33B shows the clinical scores of the differentcohorts, wherein the clinical score correlates with the survival of micein the CLP-induced sepsis model subjects. FIG. 33C shows Allocetraprevents uncontrolled cytokine signaling events, i.e., a cytokine storm,following sepsis induction, which lead to increased survival in theCLP-induced sepsis model subjects. FIG. 33D shows dose dependentincreased survival of the CLP-induced sepsis model subjects treated withAllocetra. FIG. 33E also shows dose dependent increased survival of theCLP-induced sepsis model subjects treated with Allocetra.

FIGS. 34A-34F. CLP mice display signs of respiratory and cardiovasculardysfunction that correlate with sepsis severity. (FIG. 34A) 24 hpost-CLP, naïve mice (n=21) showed no signs of illness, while themajority of CLP mice (n=40) had severe clinical signs (median MSSClinical Score of 13; 95% CI of median 9-14); **P<0.0001 by a two-tailedMann-Whitney test. (FIG. 34B) The lung-to-body weight ratiosignificantly increases with sepsis severity. (FIG. 34C) Representative2D echocardiograms of naïve (top panels) and CLP-mice (bottom panels),showing the time-lapse view (M-Mode) and top view (B-Mode). LV internaldistances, heart rate, and posterior wall thickness were measured forthe calculation of various parameters of LV structure and function,including (FIG. 34D) heart rate, (FIG. 34E) LV volume, and (FIG. 34F)cardiac output. Data are presented as the median within theinter-quartile range (IQR); mean values are marked with a ‘+’ sign;error bars represent the 5-95 percentile range; group sizes (N) areindicated below their respective legends; *P≤0.01, **P≤0.001, ***P≤0.0001 by the Kruskal-Wallis nonparametric ANOVA, with multiplecomparisons adjusted using Dunn's test. P values above the bars indicatethe significant differences from the control group, and those above thebrackets indicate the significant differences between the two othergroups.

FIGS. 35A-35C. CLP mice display signs of renal dysfunction thatcorrelate with sepsis severity. Renal dysfunction is indicated byincreasing concentrations of (FIG. 35A) blood urea, (FIG. 35B)neutrophil gelatinase-associated lipocalin (NGAL), and (FIG. 35C) serumpotassium. Data are presented as the median within the inter-quartilerange (IQR); mean values are marked with a ‘+’ sign; error barsrepresent the 5-95 percentile range; group sizes (N) are indicated belowtheir respective legends; *P≤0.01, **P≤0.001, *** P≤0.0001 by theKruskal-Wallis nonparametric ANOVA, with multiple comparisons adjustedusing Dunn's test. P values above the bars indicate differences from thecontrol group, and those above the brackets indicate differences betweenthe two other groups.

FIGS. 36A-36G. Markers for hepatic dysfunction strongly correlate withMSS clinical score in CLP mice. (FIG. 36A) 24 h post-CLP, mice withsevere sepsis (MSS Clinical Score >13) had a slight and insignificant(p>0.93) increase in total bilirubin serum concentration, while (FIG.36B) alanine aminotransferase (ALT) and (FIG. 36C) aspartateaminotransferase (AST) levels were significantly decreased with sepsisseverity. (FIG. 36D) Alkaline phosphatase and (FIG. 36E) albumin levelswere significantly decreased with sepsis severity, while (FIG. 36F)globulin serum concentrations were not significantly altered. (FIG. 36G)Glucose levels of septic mice, notably in mildly septic mice (MSSClinical score of 1-4), were lower than those in naïve mice. Data arepresented as the median within the inter-quartile range (IQR); meanvalues are marked with a ‘+’ sign; error bars represent the 5-95percentile range; group sizes (N) are indicated below their respectivelegends; *P≤0.01, **P≤0.001, *** P≤0.0001 by the Kruskal-Wallisnon-parametric ANOVA, with multiple comparisons adjusted using Dunn'stest. P values above the bars indicate the differences from the controlgroup, and those above the brackets indicate differences between the twoother groups.

FIGS. 37A-37E. Marked thrombocytopenia and lymphopenia and aberrantcomplement activation in septic mice. (FIG. 37A) 24 h post-CLP, septicmice had significantly lower platelet counts than naïve mice. Decreased(FIG. 37B) WBC and (FIG. 37C) lymphocyte counts in CLP-mice,predominantly in mice with mild sepsis (MSS clinical score of 1-4).(FIG. 37D) C5a serum concentration is higher in septic mice, regardlessof their clinical score. (FIG. 37E) C3a serum concentration is lower inseptic mice with correlation to clinical score. Data are presented asthe median within the inter-quartile range (IQR); mean values are markedwith a ‘+’ sign; error bars represent the 5-95 percentile range; groupsizes (N) are indicated below the irrespective legends; *P≤0.01,**P≤0.001, ***P≤0.0001 by the Kruskal-Wallisnon-parametric ANOVA, withmultiple-comparisons adjusted by using the Dunn's test. P values abovethe bars indicate the significant differences from the control group,and those above the brackets indicate the significant differencesbetween two other groups.

FIG. 38A-38F. CLP mice are presented with adverse metabolic changes.(FIG. 38A) 24 h after CLP, blood pH significantly decreased with sepsisseverity. (FIG. 38B) OCR measurements of PBMCs from naïve and CLP-miceshowed aberrant mitochondrial respiration, predominantly in severelyseptic mice (MSS Clinical score >10), which was manifested primarily by(FIG. 38C) a decreased coupling efficiency. (FIG. 38D) extracellularacidification rate (ECAR) measurements of PBMCs from naïve and CLP-miceshowed only mild changes in the general glycolytic function, which wasslightly increased in moderately septic mice (MSS Clinical Score 7-8.5);(FIG. 38E) The glycolytic reserve of PBMCs in this assay wassignificantly decreased in severely septic mice (MSS Clinicalscore >14). (FIG. 38F) Blood lactate concentration was slightly lower inCLP-mice. Data in FIGS. 38A, 38C, 38E, and 38F are presented as themedian within the inter-quartile range (IQR); mean values are markedwith a ‘+’ sign; error bars represent the 5-95 percentile range; data inFIGS. 38B and 38D are presented as the mean±standard deviation; groupsizes (N) are indicated below their respective legends; *P≤0.01,**P≤0.001, ***P≤0.0001 by the Kruskal-Wallis nonparametric ANOVA, withmultiple comparisons adjusted using Dunn's test. P values above the barsindicate differences from the control group, and those above thebrackets indicate differences between two other groups.

FIGS. 39A-39D. Beneficial effects of Allocetra-OTS on CLP mice. 4 hoursafter CLP, mice were injected IV with ertapenem and either Hartmann'ssolution (vehicle) or 20×10⁶ Allocetra-OTS, unless indicated otherwise.Mice were monitored for well-being and euthanized when the MSS ClinicalScore was >15. (FIG. 39A) Kaplan-Meier survival curves of CLP micetreated with either ertapenem+vehicle or ertapenem+Allocetra-OTS. (FIG.39B) Increased median survival time of Allocetra-OTS-treated mice; errorbars represent the 95% CI; *P≤0.01 by the Kruskal-Wallis nonparametricANOVA, with multiple-comparisons adjusted by using the Dunn's test.(FIG. 39C) Decreased mean MSS Clinical Score of Allocetra-OTS-treatedmice; error bars represent the standard error; ***P≤0.0001 by ordinaryone-way ANOVA of the non-linear curve fits. (FIG. 39D) Kaplan-Meiersurvival curves of CLP mice treated with ertapenem+varying doses ofAllocetra-OTS. The numbers of mice in each group (N) are indicatedbeside their respective legends.

FIGS. 40A-40L. Allocetra-OTS treatment attenuates cytokine/chemokinerelease following CLP. Blood samples were taken from C57BL/6 mice beforeCLP and 24 h, 48 h, and 72 h post-CLP, after treatment with eitherertapenem or a combination of ertapenem+Allocetra-OTS (OTS-ALC).Non-treated CLP mice did not survive past 24 h and therefore, data arenot shown. (FIGS. 40A-40L) Cytokine/chemokine levels were measured byLuminex, including: IL-6 (FIG. 40A), TNF-α (FIG. 40B), IL-11 (FIG. 40C),IL-10 (FIG. 40D), MIP-1α (FIG. 40E), MIP-1b (FIG. 40F), RANTES (FIG.40G), ENA-78 (FIG. 40H), IL-17a (FIG. 40I), IP-10 (FIG. 40J), VEGF-α(FIG. 40K), and IL-12p70 (FIG. 40L). FIGS. 40A-40L demonstrate that bothpro-inflammatory and anti-inflammatory cytokine are reduced followingtreatment of Allocetra-OTS. Data are presented as the mean±standarddeviation.

FIGS. 41A-41C. Patients Characteristics—All historical-matched controlsbetween the years 2016-2018, hospitalized in the Medical Intensive Unitat Hadassah Ein Kerem Hospital, Jerusalem, Israel, were reviewed.Historical controls were matched with patients, based on Age (±3 years),Gender matching, the Sequential Organ Failure Assessment (SOFA) score atadmission (±2), and Source of Sepsis. The probability of survival of thetreatment arm of 6 patients based on APACHE II score taken in the first24 hours of admission (a score that predicts mortality according togeneral status and chronic diseases), was 52.95%. However, no patientdied in the treated group. FIG. 41A shows the SOFA Score of patients(Treated and Controls) at the time of admission. (Black—Matchedcontrols; Light grey—Treated). FIG. 41B shows the age distribution ofpatients at the time of admission. (Black—Matched controls;Striped—Treated). FIG. 41C shows the percent of patients and theirsource of sepsis (Treated and Controls) from pneumonia, endovascularMethicillin-resistant Staphylococcus aureus (MRSA), or urinary tractinfection (UTI). The Y axis for FIGS. 41B and 41C is Percent ofpatients. The X-axis for FIG. 41B is Age.

FIG. 42 . Comparative Interim Data: Treated and Untreated MatchedControls Patient Population. FIG. 42 shows in tabular form thecomparative interim data of the six (6) treated patients andthirty-seven (37) matched control patients.

FIG. 43 . Comparative Interim Data: SOFA at Admission &Sepsis-Associated Mortality. FIG. 43 shows that for the Matched-ControlsGroup, mortalities were associated mostly with low SOFA scores atadmission.

FIGS. 44A and 44B. Comparative Interim Data: Recovery from Sepsis. FIGS.44A and 44B show that Allocetra-OTS is highly effective in treatment ofSepsis. FIG. 44A shows that 100% of all patients treated withAllocetra-OTS recovered from sepsis within 28 days, independent of thesources of sepsis, which included pneumonia, endovascularMethicillin-resistant Staphylococcus aureus (MRSA), and urinary tractinfection (UTI), compared with only 48% of matched controls. FIG. 44Bshows that 100% of sepsis patients (sources of sepsis: pneumonia andendovascular Methicillin-resistant Staphylococcus aureus (MRSA)) treatedwith Allocetra-OTS recovered from sepsis within 28 days, compared withonly 45% of matched controls. (Black—Matched Controls; LightGrey—Treated with Allocetra-OTS)

FIG. 45 . Comparative Interim Data: Recovery from Sepsis: Allocetra-OTSis highly effective in treatment of Sepsis Swiftly After Admission. FIG.45 shows % of patients having complete recovery from sepsis based on thedays from admission, wherein 100% of treated patients (dark grey line)recovered by day 8, compared with less than 40% of matched controls(light grey line).

FIGS. 46A and 46B. Comparative Top-Line Data: Recovery from Sepsis:Mortality Data showing that Allocetra-OTS is highly effective inprevention of Sepsis-associated mortality. FIG. 46A shows no deaths ofAllocetra treated patients, independent of the source of sepsis, whichincluded pneumonia, endovascular Methicillin-resistant Staphylococcusaureus (MRSA), and urinary tract infection (UTI), compared withmortality rates of 29% (matched controls) and 23% (literature). FIG. 46Bshows no deaths of Allocetra treated patients, when the source of sepsisincluded pneumonia and endovascular Methicillin-resistant Staphylococcusaureus (MRSA), compared with mortality rates of 34% (matched controls)and 28% (literature). (Black—Matched Controls; Light Grey—Literature)

FIGS. 47A and 47B. Comparative Interim Data: Duration of ICUHospitalization showing that Allocetra-OTS improves patients' clinicalstate and speeds up release from ICU. FIG. 47A shows that after 6 days,only 43% of all Matched Controls were released from the ICU, comparedwith 100% of patients treated with Allocetra-OTS. (Treated—dashed blacklines; Matched Control—dotted lines) FIG. 47B shows that after 6 days,only 35% of Matched Controls (Excluding UTI controls) were released fromthe ICU, compared with 100% of patients treated with Allocetra-OTS.(Treated—dashed black lines; Matched Control—solid grey lines; Datacollection started at 100%)

FIGS. 48A-48D. Comparative Interim Data: Organ Dysfunction & Failureshowing that Allocetra-OTS prevents organ dysfunction and failure. FIG.48A shows the average baseline SOFA and the maximum SOFA for allsubjects, independent of the source of sepsis, which included pneumonia,endovascular Methicillin-resistant Staphylococcus aureus (MRSA), andurinary tract infection (UTI) (Treated and Matched Controls). FIG. 48Bshows the average baseline SOFA and the maximum SOFA for subjects(Treated and Matched Controls) excluding the UTI patients. FIG. 48Cshows the median baseline SOFA and the maximum SOFA for all subjects,independent of the source of sepsis, which included pneumonia,endovascular Methicillin-resistant Staphylococcus aureus (MRSA), andurinary tract infection (UTI) (Treated and Matched Controls). There wasno difference between treated patients and historical controls at thebaseline SOFA but there was a dramatic difference between maximal SOFA,indicating that treated patients did not progress in their sepsiscourse. FIG. 48D shows the median baseline SOFA and the maximum SOFA forsubjects (Treated and Matched Controls) excluding the UTI patients.(Black—Matched Controls; Light Grey—Treated with Allocetra-OTS)

FIGS. 49A and 49B. Comparative Interim Data: Organ Dysfunction & Failureshowing that treatment with Allocetra-OTS prevented an increase in SOFAscores. FIG. 49A shows the percent of all patients with increased SOFAscores from time of admission, independent of the source of sepsis,which included pneumonia, endovascular Methicillin-resistantStaphylococcus aureus (MRSA), and urinary tract infection (UTI). FIG.49B shows the percent of patients with increased SOFA scores from timeof admission, wherein the source of sepsis included pneumonia andendovascular Methicillin-resistant Staphylococcus aureus (MRSA).(Black—Matched Controls; Light Grey—Allocetra-OTS Treated)

FIGS. 50A and 50B. Comparative Interim Data: Organ Dysfunction & Failureshowing that prevention of SOFA increase by 4 or more points is criticalto prevent mortality and that treatment with Allocetra-OTS prevents SOFAincrease by 4 or more points. FIG. 50A shows the percent of mortality ofpatients with a SOFA increase of greater than or equal to 4. FIG. 50Bshows the percent of patients with a SOFA increase of greater than orequal to 4. (Black—Matched Controls; Light Grey—Allocetra-OTS Treated)

FIGS. 51A-51D. Comparative Final Data: WBC count (FIG. 51A), Neutrophilcount (FIG. 51B), Lymphocyte count (FIG. 51C) and C-reactive protein(CRP) levels (FIG. 51D) in 10 patients as a function of time postscreening.

FIGS. 52A and 52B. Comparative Final Data: Survival of entire patientpopulation (10 patients, (FIG. 52A) and pneumonia patients only (FIG.52B) as a function of follow up time.

FIGS. 53A and 53B. Comparative Final Data: Time to discharge fromgeneral Hospital (FIG. 53A) and from ICU (FIG. 5B) as a function of timepost admission.

FIGS. 54A, 54B, 54C, and 54D show pro- and anti-inflammatory cytokine,growth factor, chemokine, immune modulator, and endocrine hormonekinetics during sepsis. Comparative Final Data: FIG. 554A showsresolution of the cytokine storm in sepsis following administration ofAllocetra-OTS (early apoptotic cells). Shown here are embodiments ofpro-inflammatory cytokines levels (IL-6, TNF-alpha, IL-1beta, IL-18,IFN-gamma). Pro-inflammatory cytokine kinetics during sepsis. Serum wasobtained from patients at the indicated times and cytokine analysis wasperformed as described in Methods. The serum of three healthy volunteerswas analyzed using the same methods and is presented as the normal range(median±range). Serum concentrations of IL-6, TNF-α, IL-1β, IL-18, andIFN-γ are presented. Patients 01-08 received one dose of Allocetra-OTSon day 1 and patients 09-12 received two doses on days 1 and 3. FIG. 54Bpresents resolution of the cytokine storm in sepsis followingadministration of Allocetra-OTS (early apoptotic cells). Shown here areembodiments of anti-inflammatory cytokines levels and levels ofhemopoietic growth factors (IL-10, IL1Ra, TNFR-1, G-CSF, VEGF, GM-CSF).Anti-Inflammatory cytokine and growth factor kinetics during sepsis.Serum was obtained from patients at the indicated times andcytokine/growth factor analysis was performed as described in Methods.The serum of three healthy volunteers was analyzed using the samemethods and is presented as the normal range (median±range). Serumconcentrations of IL-10, IL-1Ra, TNF-R1, G-CSF, VEGF, and GM-CSF, are.Patients 01-08 received one dose of Allocetra-OTS on day 1 and patients09-12 received two doses on days 1 and 3. FIG. 54C presents resolutionof the cytokine storm in sepsis following administration ofAllocetra-OTS (early apoptotic cells). Shown here are embodiments ofchemokine levels (MCP-1, IP-10, MOP-1alpha, IL-8, Gro-8, RANTES,Cortisol, FT3). Chemokine kinetics during sepsis. Serum was obtainedfrom patients at the indicated times and cytokine/growth factorsanalysis was performed as described in Methods. The serum of threehealthy volunteers was analyzed using the same methods and is presentedas the normal range (median±range). Serum concentrations of MCP-1,IP-10, MIP-1α, IL-8, GRO-□, and RANTES, are presented. Patients 01-08received one dose of Allocetra-OTS on day 1 and patients 09-12 receivedtwo doses on days 1 and 3. FIG. 54D presents resolution of the cytokinestorm in sepsis following administration of Allocetra-OTS (earlyapoptotic cells). Shown here are embodiments of immuno-modulatingfactors levels (TREM-1, Osteopontin, NGAL, Ghrelin, Leptin, Glucagon).Immune modulator and endocrine hormone kinetics during sepsis. Serum wasobtained from patients at the indicated times and cytokine/growthfactors analysis was performed as described in Methods. The serum ofthree healthy volunteers was analyzed using the same methods and ispresented as the normal range (median±range). Serum concentrations of(A) TREM-1, (B) osteopontin, (C) N-GAL, (D) ghrelin, (E) leptin, and (F)glucagon, (G) cortisol, and (H) FT3, are presented. Patients 01-08received one dose of Allocetra-OTS on day 1 and patients 09-12 receivedtwo doses on days 1 and 3.

FIGS. 55A, 55B, 55C, and 55D. Comparative Final Data: FIG. 55A showsaverage delta SOFA scores (between day 1-5 (Left-hand side—Delta SOFAscore before administration of early apoptotic cells (Allocetra-OTS) andat day 5; p<0.0001 T-test), day 1-7 (Middle—Delta SOFA score beforeadministration of early apoptotic cells (Allocetra-OTS) and at day 7;p<0.0001 T-test), and day 1-28 (Right-hand side—Delta SOFA score beforeadministration of early apoptotic cells (Allocetra-OTS) and at day 7;p<0.0005 T-test)); FIG. 55B shows Sequential Organ Failure Assessment(SOFA) score progression during sepsis-AUC of SOFA between day 1-5(Left-hand side; p<0.0001 T-test) and day 1-7 (Right-hand side; p<0.0001T-test); FIG. 55C shows maximal SOFA score; and FIG. 55D shows durationof stay in the hospital and in ICU/IMU (all patients in the study).

DETAILED DESCRIPTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the methodsdisclosed herein. However, it will be understood by those skilled in theart that these methods may be practiced without these specific details.In other instances, well-known methods, procedures, and components havenot been described in detail so as not to obscure the methods disclosedherein.

Genetic modification of immune cells is well known as a strategy forimmune-cell therapies against cancer. These immune-cell therapies arebased on the manipulation and administration of autologous or allogeneicimmune cells to a subject in need. Immune-cell based therapies includenatural killer cells therapies, dendrite cell therapies, and T-cellimmunotherapies including those utilizing naïve T-cells, effectorT-cells also known as T-helper cells, cytotoxic T-cells, and regulatoryT-cells (Tregs).

In some embodiments, disclosed herein are compositions comprisinggenetically modified immune cells. In another embodiment, thegenetically modified immune cell is a T-cell. In another embodiment, aT-cell is a naïve T-cell. In another embodiment, a T-cell is a naïveCD4⁺ T-cell. In another embodiment, a T-cell is a naïve T-cell. Inanother embodiment, a T-cell is a naïve CD8⁺ T-cell. In anotherembodiment, the genetically modified immune cell is a natural killer(NK) cell. In another embodiment, the genetically modified immune cellis a dendritic cell. In still another embodiment, the geneticallymodified T-cell is a cytotoxic T lymphocyte (CTL cell). In anotherembodiment, the genetically modified T-cell is a regulatory T-cell(Treg). In another embodiment, the genetically modified T-cell is achimeric antigen receptor (CAR) T-cell. In another embodiment, thegenetically modified T-cell is a genetically modified T-cell receptor(TCR) cell.

In some embodiments, disclosed herein are compositions comprisinggenetically modified immune cells and apoptotic cells. In anotherembodiment, disclosed herein are compositions comprising geneticallymodified immune cells and supernatants from apoptotic cells. In anotherembodiment, the genetically modified immune cell is a T-cell. In anotherembodiment, the genetically modified immune cell is a natural killer(NK) cell. In still another embodiment, the genetically modified immunecell is a cytotoxic T lymphocyte (CTL cell). In another embodiment, thegenetically modified immune cell is a regulatory T lymphocyte (Tregcell).

In some embodiments, disclosed herein is a method of maintaining orincreasing the proliferation rate of chimeric antigenreceptor-expressing T-cells (CAR T-cell) during CAR T-cell cancertherapy, the method comprising the step of administering a compositioncomprising apoptotic cells or an apoptotic cell supernatant to saidsubject, and wherein said proliferation rate is maintained or increasedin the subject compared with a subject undergoing CAR T-cell cancertherapy and not administered said apoptotic cells or said apoptotic cellsupernatant.

In a related embodiment, the method does not reduce or inhibit theefficacy of said CAR T-cell cancer therapy. In a related embodiment, themethod improves the efficacy of said CAR T-cell cancer therapy. Inanother related embodiment the incidence of cytokine release syndrome(CRS) or a cytokine storm in said subject is inhibited or reducedcompared with a subject not administered said apoptotic cells or saidapoptotic cell supernatant.

In some embodiments, CRS occurs spontaneously. In another embodiment,CRS occurs in response to LPS. In another embodiment, CRS occurs inresponse to IFN-γ.

In some embodiments, disclosed herein is a method of increasing theefficacy of chimeric antigen receptor T-cell (CAR T-cell) cancertherapy, the method comprising the step of administering CAR T-cells andan additional agent selected from the group comprising apoptotic cells,an apoptotic cell supernatant, a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, a tellurium-basedcompound, or an immune modulating agent, or any combination thereof,wherein said efficacy said CAR T-cells is increased in the subjectcompared with a subject undergoing CAR T-cell cancer therapy and notadministered said additional agent. In a related embodiment, the levelof production of at least one pro-inflammatory cytokine is reducedcompared with the level of said pro-inflammatory cytokine in a subjectreceived CAR T-cell cancer therapy and not administered a compositioncomprising said agent. In another related embodiment, thepro-inflammatory cytokine comprises IL-6.

In a related embodiment, when apoptotic cells or an apoptotic cellsupernatant is administered, said method increases the levels of IL-2 inthe subject compared with a subject undergoing CAR T-cell cancer therapyand not administered said apoptotic cells or said apoptotic cellsupernatant. In another embodiment, when apoptotic cells or an apoptoticcell supernatant is administered, said method maintains the levels ofIL-2 in the subject compared with a subject undergoing CAR T-cell cancertherapy and not administered said apoptotic cells or said apoptotic cellsupernatant. In another embodiment, when apoptotic cells or an apoptoticcell supernatant is administered, said method maintains or increases thelevels of IL-2 in the subject compared with a subject undergoing CART-cell cancer therapy and not administered said apoptotic cells or saidapoptotic cell supernatant. In another related embodiment, the incidenceof cytokine release syndrome (CRS) or a cytokine storm in said subjectis inhibited or reduced compared with a subject not administered saidadditional agent.

In a related embodiment, CAR T-cells and said additional agent or anycombination thereof are comprised in a single composition. In anotherrelated embodiment, said CAR T-cell and said additional agent or anycombination thereof are comprised in at least two compositions.

In some embodiments, disclosed herein is a method of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor in a subject, comprising the step ofadministering chimeric antigen receptor-expressing T-cells (CAR T-cell)and an additional agent, said additional agent comprising apoptoticcells, apoptotic supernatants or a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, a tellurium-basedcompound, or an immune modulating agent, or any combination thereof,wherein said method treats, prevents, inhibits, reduces the incidenceof, ameliorates or alleviates a cancer or a tumor in said subjectcompared with a subject administered CAR T-cells and not administeredsaid additional agent.

In a related embodiment, said method has increased efficacy treating,preventing, inhibiting, reducing the incidence of, ameliorating oralleviating said cancer or said tumor in said subject compared with asubject administered CAR T-cells and not administered said additionalagent.

In another related embodiment, the level of production of at least onepro-inflammatory cytokine is reduced compared with the level of saidpro-inflammatory cytokine in a subject administered said CAR T-cells andnot administered a composition comprising said agent. In another relatedembodiment, said pro-inflammatory cytokine comprises IL-6. In anotherrelated embodiment, said additional agent comprises apoptotic cells oran apoptotic cell supernatant, said method increases the levels of IL-2in the subject compared with a subject administered said CAR T-cells andnot administered said apoptotic cells or said apoptotic cellsupernatant. In another related embodiment, said CAR T-cells and saidadditional agent or any combination thereof are comprised in a singlecomposition. In yet another related embodiment, said CAR T-cells andsaid additional agent or any combination thereof are comprised in atleast two compositions.

In a related embodiment, the administration of said additional agentoccurs prior to, concurrent with, or following the administration ofsaid CAR T-cells. In another related embodiment, said apoptotic cellscomprise apoptotic cells in an early-apoptotic state. In another relatedembodiment, said apoptotic cells are autologous to said subject or arepooled third-party donor cells.

In a related embodiment, said apoptotic cell supernatant is obtained bya method comprising the steps of (a) providing apoptotic cells, (b)culturing the cells of step (a), and (c) separating the supernatant fromthe cells. In another related embodiment, said apoptotic cellsupernatant is an apoptotic cell-white blood cell supernatant and saidmethod further comprises the steps of: (d) providing white blood cells,(e) optionally, washing the apoptotic cells and the white blood cells,(f) co-culturing the apoptotic cells and the white blood cells, whereinsteps (d)-(f) are in place of step (b). In another related embodiment,the provided white blood cells are selected from the group consisting ofphagocytes, macrophages, dendritic cells, monocytes, B cells, T cells,and NK cells. Thus, in some embodiments, apoptotic supernatants comprisea supernatant produced by culturing apoptotic cells with macrophages,wherein the macrophage ingests the apoptotic cells and the supernatantproduced from this co-culturing is used. In some embodiments, apoptoticsupernatants comprise a supernatant produced by culturing apoptoticcells, wherein the supernatant is produced from materials secreted bythe apoptotic cells.

In some embodiments, disclosed herein are compositions comprising earlyapoptotic cells. In some embodiments, disclosed herein are compositionscomprising early apoptotic cells in combination with an additionalagent. In some embodiments, the additional agent may be a CAR T-cell. Insome embodiments, the additional agent may be an antibody. In someembodiments, the antibody comprises rituximab or a functional fragmentthereof.

In some embodiments, compositions of early apoptotic cells comprise apopulation of mononuclear apoptotic cell comprising mononuclear cells inan early-apoptotic state, wherein said mononuclear apoptotic cellpopulation comprises: a decreased percent of non-quiescent non-apoptoticviable cells; a suppressed cellular activation of any livingnon-apoptotic cells; or a reduced proliferation of any livingnon-apoptotic cells; or any combination thereof.

In some embodiments, disclosed herein are compositions comprisinggenetically modified T-cells and apoptotic cells. In another embodiment,disclosed herein are compositions comprising genetically modifiedT-cells and supernatants of apoptotic cells. In another embodiment, thegenetically modified T-cell is a chimeric antigen receptor (CAR) T-cell.In another embodiment, the genetically modified T-cell is a geneticallymodified T-cell receptor (TCR) cell.

In some embodiments, disclosed herein are compositions comprising CART-cells and apoptotic cells. In another embodiment, disclosed herein arecompositions comprising genetically modified T-cell receptor cells(TCRs) and apoptotic cells. In another embodiment, disclosed herein arecompositions comprising CAR T-cells and supernatants from apoptoticcells. In another embodiment, disclosed herein are compositionscomprising genetically modified T-cell receptor cells (TCRs) andsupernatant of apoptotic cells.

In certain embodiments, genetically modified immune cells and apoptoticcells or apoptotic cell supernatants are comprised within a singlecomposition. In other embodiments, genetically modified immune cells andapoptotic cells or apoptotic cell supernatants are comprised in separatecompositions.

This disclosure provides in some embodiments, a pooled mononuclearapoptotic cell preparation comprising mononuclear cells in an earlyapoptotic state, wherein said pooled mononuclear apoptotic cellspreparation comprises pooled individual mononuclear cell populations,and wherein said pooled mononuclear apoptotic cell preparation comprisesa decreased percent of living non-apoptotic cells, a suppressed cellularactivation of any living non-apoptotic cells, or a reduced proliferationof any living non-apoptotic cells, or any combination thereof. Inanother embodiment, the pooled mononuclear apoptotic cells have beenirradiated. In another embodiment, this disclosure provides a pooledmononuclear apoptotic cell preparation that in some embodiments, usesthe white blood cell fraction (WBC) obtained from donated blood. Oftenthis WBC fraction is discarded at blood banks or is targeted for use inresearch.

In some embodiments, a cell population disclosed herein is inactivated.In another embodiment, inactivation comprises irradiation. In anotherembodiment, inactivation comprises T-cell receptor inactivation. Inanother embodiment, inactivation comprises T-cell receptor editing. Inanother embodiment, inactivation comprises suppressing or eliminating animmune response in said preparation. In another embodiment, inactivationcomprises suppressing or eliminating cross-reactivity between multipleindividual populations comprised in the preparation. In otherembodiment, inactivation comprises reducing or eliminating T-cellreceptor activity between multiple individual populations comprised inthe preparation. In another embodiment, an inactivated cell preparationcomprises a decreased percent of living non-apoptotic cells, suppressedcellular activation of any living non-apoptotic cells, or a reduceproliferation of any living non-apoptotic cells, or any combinationthereof.

In another embodiment, an inactivated cell population comprises areduced number of non-quiescent non-apoptotic cells compared with anon-radiated cell preparation. In some embodiments, an inactivated cellpopulation comprises 50 percent (%) of living non-apoptotic cells. Insome embodiments, an inactivated cell population comprises 40% of livingnon-apoptotic cells. In some embodiments, an inactivated cell populationcomprises 30% of living non-apoptotic cells. In some embodiments, aninactivated cell population comprises 20% of living non-apoptotic cells.In some embodiments, an inactivated cell population comprises 100% ofliving non-apoptotic cells. In some embodiments, an inactivated cellpopulation comprises 0% of living non-apoptotic cells.

In some embodiments, disclosed herein is a method of preparing aninactivated early apoptotic cell population. In some embodiments,disclosed herein is a method for producing a population of mononuclearapoptotic cell comprising a decreased percent of non-quiescentnon-apoptotic viable cells; a suppressed cellular activation of anyliving non-apoptotic cells; or a reduced proliferation of any livingnon-apoptotic cells; or any combination thereof, said method comprisingthe following steps,

obtaining a mononuclear-enriched cell population of peripheral blood;

freezing said mononuclear-enriched cell population in a freezing mediumcomprising an anticoagulant;

thawing said mononuclear-enriched cell population;

incubating said mononuclear-enriched cell population in an apoptosisinducing incubation medium comprising methylprednisolone at a finalconcentration of about 10-100 μg/mL and an anticoagulant;

resuspending said apoptotic cell population in an administration medium;and inactivating said mononuclear-enriched population, wherein saidinactivation occurs following induction,

wherein said method produces a population of mononuclear apoptotic cellcomprising a decreased percent of non-quiescent non-apoptotic cells; asuppressed cellular activation of any living non-apoptotic cells; or areduced proliferation of any living non-apoptotic cells; or anycombination thereof.

In another embodiment, the irradiation comprises gamma irradiation or UVirradiation. In yet another embodiment, the irradiated preparation has areduced number of non-quiescent non-apoptotic cells compared with anon-irradiated cell preparation.

In another embodiment, the pooled mononuclear apoptotic cells haveundergone T-cell receptor inactivation. In another embodiment, thepooled mononuclear apoptotic cells have undergone T-cell receptorediting.

In some embodiments, pooled blood comprises 3^(rd) party blood from HLAmatched or HLA unmatched sources, with respect to a recipient.

In some embodiments, disclosed herein are compositions comprisinggenetically modified immune cells, for example but not limited to CART-cells and an additional agent selected from the group comprisingapoptotic cells, an apoptotic cell supernatant, a CTLA-4 blocking agent,an alpha-1 anti-trypsin or fragment thereof or analogue thereof, atellurium-based compound, or an immune modulating agent, or anycombination thereof.

In some embodiments, this disclosure provides methods of production of apharmaceutical composition comprising a pooled mononuclear apoptoticcell preparation comprising pooled individual mononuclear cellpopulations in an early apoptotic state, wherein said compositioncomprises a decreased percent of living non-apoptotic cells, apreparation having a suppressed cellular activation of any livingnon-apoptotic cells, or a preparation having reduced proliferation ofany living non-apoptotic cells, or any combination thereof. In anotherembodiment, the methods provide a pharmaceutical composition comprisinga pooled mononuclear apoptotic cell preparation comprising pooledindividual mononuclear cell populations in an early apoptotic state,wherein said composition comprises a decreased percent of non-quiescentnon-apoptotic cells.

In some embodiments, disclosed herein is a method of treating,preventing, inhibiting the growth of, reducing the incidence of, or anycombination thereof, a cancer or a tumor in a subject, comprising a stepof administering an early apoptotic cell population to said subject,wherein said method treats, prevents, inhibits the growth of, reducesthe incidence of, or any combination thereof, a cancer or a tumor insaid subject. In some embodiments, methods herein comprise treating,preventing, inhibiting the growth of, delaying disease progression,reducing the tumor load, or reducing the incidence of a cancer or atumor in a subject, or any combination thereof, comprising a step ofadministering a composition comprising an early apoptotic cellpopulation to said subject. In some embodiments, the method furthercomprises administering an additional immune therapy, a chemotherapeuticagent, or an immune modulator to said subject, or any combinationthereof. In some embodiments, the additional immune therapy, achemotherapeutic agent, or an immune modulator is administered prior to,concurrent with, or following administration of said early apoptoticcells.

In some embodiments, disclosed herein is a method of increasing survivalof a subject suffering from a cancer or a tumor, comprising a step ofadministering an early apoptotic cell population to said subject,wherein said method increases survival of said subject. In someembodiments, the method further comprises administering an additionalimmune therapy, a chemotherapeutic agent, or an immune modulator to saidsubject, or any combination thereof. In some embodiments, the additionalimmune therapy, a chemotherapeutic agent, or an immune modulator isadministered prior to, concurrent with, or following administration ofsaid early apoptotic cells.

In some embodiments, disclosed herein is a method of reducing the sizeor reducing the growth rate of a cancer or a tumor, or a combinationthereof, in a subject, comprising a step of administering an earlyapoptotic cell population to said subject, wherein said method reducesthe size or reduces the growth rate. In some embodiments, the methodfurther comprises administering an additional immune therapy, achemotherapeutic agent, or an immune modulator to said subject, or anycombination thereof. In some embodiments, the additional immune therapy,a chemotherapeutic agent, or an immune modulator is administered priorto, concurrent with, or following administration of said early apoptoticcells.

In some embodiments, administration of a composition comprisingapoptotic cells does not affect the efficacy of CAR T-cells to treat,prevent, inhibit, reduce the incidence of, ameliorating, reduce thetumor load, or alleviating a cancer or a tumor. In another embodiment,administration of a composition comprising apoptotic cells does notreduce the efficacy of the CAR T-cells to treat, prevent, inhibit,reduce the incidence of, ameliorating, reduce the tumor load, oralleviating a cancer or a tumor by more than about 5%. In anotherembodiment, administration of a composition comprising apoptotic cellsdoes not reduce the efficacy of the CAR T-cells to treat, prevent,inhibit, reduce the incidence of, ameliorating, reduce the tumor load,or alleviating a cancer or a tumor by more than about 10%. In anotherembodiment, administration of a composition comprising apoptotic cellsdoes not reduce the efficacy of the CAR T-cells to treat, prevent,inhibit, reduce the incidence of, ameliorating, reduce the tumor load,or alleviating a cancer or a tumor by more than about 15%. In anotherembodiment, administration of a composition comprising apoptotic cellsdoes not reduce the efficacy of the CAR T-cells to treat, prevent,inhibit, reduce the incidence of, ameliorating, reduce the tumor load,or alleviating a cancer or a tumor by more than about 20%.

In some embodiments, administration of apoptotic cells increases theefficacy of CAR T-cells. In some embodiments, administration ofapoptotic cells increases the efficacy of CAR T-cells by at least 5, byat least 10%, by at least 15%, by at least 20%, by at least 25, by atleast 30%, by at least 35%, by at least 40%, by at least 45, or by atleast 50%.

In another embodiment, administration of a composition comprising anapoptotic cell supernatant does not reduce the efficacy of the CART-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 5%. In another embodiment, administration of a compositioncomprising an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 10%. In another embodiment, administration of a compositioncomprising an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 15%. In another embodiment, administration of a compositioncomprising an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor by more thanabout 20%. In another embodiment, administration of a compositioncomprising the apoptotic cell supernatant does not affect the efficacyof the CAR T-cells to treat, prevent, inhibit, reduce the incidence of,ameliorating, or alleviating said cancer or said tumor. In anotherembodiment, administration of a composition comprising the apoptoticcell supernatant does not reduce the efficacy of the CAR T-cells totreat, prevent, inhibit, reduce the incidence of, ameliorating, oralleviating said cancer or said tumor.

In some embodiments, disclosed herein are methods of inhibiting orreducing the incidence of cytokine release syndrome (CRS) or cytokinestorm in a subject undergoing CAR T-cell cancer therapy. In anotherembodiment, methods disclosed herein decrease or prevent cytokineproduction in a subject undergoing CAR T-cell cancer therapy therebyinhibiting or reducing the incidence of cytokine release syndrome (CRS)or cytokine storm in a subject. In another embodiment, the methodsdisclosed herein of inhibiting or reducing the incidence of cytokinerelease syndrome (CRS) or cytokine storm in a subject undergoing CART-cell cancer therapy comprise the step of administering a compositioncomprising apoptotic cells to the subject undergoing the cancer therapy.In yet another embodiment, methods disclosed herein for decreasing orinhibiting cytokine production in a subject undergoing CAR T-cell cancertherapy comprise the step of administering a composition comprisingapoptotic cells to the subject undergoing the cancer therapy. In anotherembodiment, administration of a composition comprising apoptotic cellsdoes not affect the efficacy of the CAR T-cell therapy. In anotherembodiment, administration of a composition comprising apoptotic cellsor an apoptotic supernatant does not reduce the efficacy of the CART-cell therapy. In another embodiment, administration of a compositioncomprising apoptotic cells or an apoptotic cell supernatant does notreduce the efficacy of the CAR T-cells therapy by more than about 5%. Inanother embodiment, administration of a composition comprising apoptoticcells or an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cells therapy by more than about 10%. In another embodiment,administration of a composition comprising apoptotic cells or anapoptotic cell supernatant does not reduce the efficacy of the CART-cells therapy by more than about 15%. In another embodiment,administration of a composition comprising apoptotic cells or anapoptotic cell supernatant does not reduce the efficacy of the CART-cells therapy by more than about 20%.

In some embodiments, disclosed herein are methods of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm comprising the step of administering anapoptotic cell supernatant, as disclosed herein, or a compositioncomprising said apoptotic cell supernatant. In another embodiment, anapoptotic cell supernatant comprises an apoptotic cell-phagocytesupernatant.

In some embodiments, methods disclosed herein for decreasing orinhibiting cytokine production in a subject undergoing CAR T-cell cancertherapy comprise the step of administering a composition comprising anapoptotic cell supernatant to the subject undergoing the cancer therapy.In another embodiment, administration of a composition comprising anapoptotic cell supernatant does not affect the efficacy of the CART-cell therapy. In another embodiment, administration of a compositioncomprising an apoptotic cell supernatant does not reduce the efficacy ofthe CAR T-cell therapy.

In some embodiments, a method of inhibiting or reducing the incidence ofa cytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing chimeric antigen receptor-expressing T-cell (CAR T-cell)cancer therapy comprises the step of administering a compositioncomprising apoptotic cells or an apoptotic supernatant to said subject.In another embodiment, a method of inhibiting or reducing the incidenceof a cytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing chimeric antigen receptor-expressing T-cell (CAR T-cell)cancer therapy decreases or inhibits production of at least onepro-inflammatory cytokine in the subject.

In another embodiment, this disclosure provides methods of use of apooled mononuclear apoptotic cell preparation comprising mononuclearcells in an early apoptotic state, as described herein, for treating,preventing, ameliorating, inhibiting, or reducing the incidence of animmune disease, an autoimmune disease, an inflammatory disease, acytokine release syndrome (CRS), a cytokine storm, or infertility in asubject in need thereof. In another embodiment, disclosed herein is apooled mononuclear apoptotic cell preparation, wherein use of such acell preparation in certain embodiments does not require matching donorsand recipients, for example by HLA typing.

Genetically Modified Immune Cells

Genetic modification of immune cells is well known as a strategy forimmune-cell therapies against cancer. These immune-cell therapies arebased on the manipulation and administration of autologous or allogeneicimmune cells to a subject in need. Immune-cell based therapies includenatural killer cells therapies, dendrite cell therapies, and T-cellimmunotherapies including those utilizing naïve T-cells, effectorT-cells also known as T-helper cells, cytotoxic T-cells, and regulatoryT-cells (Tregs).

In one embodiment, disclosed herein are compositions comprisinggenetically modified immune cells In another embodiment, the geneticallymodified immune cell is a T-cell. In another embodiment, a T-cell is anaïve T-cell. In another embodiment, a T-cell is a naïve CD4⁺ T-cell. Inanother embodiment, a T-cell is a naïve T-cell. In another embodiment, aT-cell is a naïve CD8⁺ T-cell. In another embodiment, the geneticallymodified immune cell is a natural killer (NK) cell. In anotherembodiment, the genetically modified immune cell is a dendritic cell. Instill another embodiment, the genetically modified T-cell is a cytotoxicT lymphocyte (CTL cell). In another embodiment, the genetically modifiedT-cell is a regulatory T-cell (Treg). In another embodiment, thegenetically modified T-cell is a chimeric antigen receptor (CAR) T-cell.In another embodiment, the genetically modified T-cell is a geneticallymodified T-cell receptor (TCR) cell.

In one embodiment, disclosed herein are compositions comprisinggenetically modified immune cells and an additional agent selected fromthe group comprising apoptotic cells, an apoptotic cell supernatant, aCTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereof oranalogue thereof, a tellurium-based compound, or an immune modulatingagent, or any combination thereof. In another embodiment, disclosedherein are compositions comprising genetically modified immune cells,apoptotic cells, and an additional agent selected from the groupcomprising a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragmentthereof or analogue thereof, a tellurium-based compound, or an immunemodulating agent, or any combination thereof. In another embodiment,disclosed herein are compositions comprising genetically modified immunecells, an apoptotic cell supernatant, and an additional agent selectedfrom the group comprising a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, a tellurium-basedcompound, or an immune modulating agent, or any combination thereof.

In one embodiment, the immune cells are cytotoxic. In anotherembodiment, cytotoxic cells for genetic modification can be obtainedfrom bone marrow of the subject (autologous) or a donor (allogeneic). Inother cases, the cells are obtained from a stem cell. For example,cytotoxic cells can be derived from human pluripotent stem cells such ashuman embryonic stem cells or human induced pluripotent T-cells. In thecase of induced pluripotent stem cells (IPSCs), such pluripotent T-cellscan be obtained using a somatic cell from the subject to whichgenetically modified cytotoxic cells will be provided. In oneembodiment, immune cells may be obtained from a subject or donor byharvesting cells by venipuncture, by apheresis methods, by white cellmobilization followed by apheresis or venipuncture, or by bone marrowaspiration.

In one embodiment, immune cells, for example T-cell, are generated andexpanded by the presence of specific factors in vivo. In anotherembodiment, T-cell generation and maintenance is affected by cytokinesin vivo. In another embodiment, cytokines that affect generation andmaintenance to T-helper cells in vivo comprise IL-1, IL-2, IL-4, IL-6,IL-12, IL-21, IL-23, IL-25, IL-33, and TGFβ. In another embodiment, Tregcells are generated from naïve T-cells by cytokine induction in vivo. Instill another embodiment, TGF-β and/or IL-2 play a role indifferentiating naïve T-cell to become Treg cells.

In another embodiment, the presence of a cytokine selected from thegroup comprising IL-1, IL-2, IL-4, IL-6, IL-12, IL-21, IL-23, IL-25,IL-33, and TGFβ, maintains or increases the proliferation rate or both,of T-cells in vivo. In another embodiment, the presence of a cytokineIL-2 and/or TGFβ, maintains or increases the proliferation rate or both,of T-cells in vivo. In another embodiment, the presence of a cytokineselected from the group comprising IL-1, IL-2, IL-4, IL-6, IL-12, IL-21,IL-23, IL-25, IL-33, and TGFβ, maintains or increases the proliferationrate or both, of CAR T-cells in vivo. In another embodiment, thepresence of a cytokine IL-2 and/or TGFβ, maintains or increases theproliferation rate or both, of CAR T-cells in vivo. In anotherembodiment, the presence of a cytokine selected from the groupcomprising IL-1, IL-2, IL-4, IL-6, IL-12, IL-21, IL-23, IL-25, IL-33,and TGFβ, maintains or increases the proliferation rate or both, of TCRT-cells in vivo. In another embodiment, the presence of a cytokine IL-2and/or TGFβ, maintains or increases the proliferation rate or both, ofTCR T-cells in vivo. In another embodiment, the presence of a cytokineselected from the group comprising IL-1, IL-2, IL-4, IL-6, IL-12, IL-21,IL-23, IL-25, IL-33, and TGFβ, maintains or increases the proliferationrate or both, of T-reg cells in vivo. In another embodiment, thepresence of a cytokine IL-2 and/or TGFβ, maintains or increases theproliferation rate or both, of T-reg cells in vivo.

In one embodiment T-cells having an altered expression or form of STAT5Bencoded protein or BACH2 encoded protein are maintained for an extendedtime period or have an increased proliferation rate or both. In anotherembodiment, said altered expression increases expression STAT5Bpolypeptide. In another embodiment, said altered expression increasesexpression of BACH2 polypeptide.

In another embodiment, T-cells having an altered expression of a STAT5Bencoded protein are maintained for an extended time period or have anincreased proliferation rate in vivo. In another embodiment, T-cellshaving an altered expression of a BACH2 encoded protein are maintainedfor an extended time period or have an increased proliferation rate invivo. In another embodiment, T-cells having an altered form of a STAT5Bencoded protein are maintained for an extended time period or have anincreased proliferation rate in vivo. In another embodiment, T-cellshaving an altered form of a BACH2 encoded protein are maintained for anextended time period or have an increased proliferation rate in vivo.

In another embodiment, T-cells having an altered expression of a STAT5Bencoded protein maintain or increase their proliferation rate in vivofor greater than 1 year. In another embodiment, T-cells having analtered expression of a STAT5B encoded protein maintain or increasetheir proliferation rate in vivo for greater than 2 years. In anotherembodiment, T-cells having an altered expression of a STAT5B encodedprotein maintain or increase their proliferation rate in vivo forgreater than 3 years. In another embodiment, T-cells having an alteredexpression of a STAT5B encoded protein maintain or increase theirproliferation rate in vivo for greater than 4 years. In anotherembodiment, T-cells having an altered expression of a STAT5B encodedprotein maintain or increase their proliferation rate in vivo forgreater than 5 years. In another embodiment, T-cells having an alteredexpression of a STAT5B encoded protein maintain or increase theirproliferation rate in vivo for greater than 10 years. In anotherembodiment, T-cells having an altered expression of a STAT5B encodedprotein maintain or increase their proliferation rate in vivo forgreater than 20 years.

In another embodiment, T-cells having an altered expression of a BACH2encoded protein maintain or increase their proliferation rate in vivofor greater than 1 year. In another embodiment, T-cells having analtered expression of a BACH2 encoded protein maintain or increase theirproliferation rate in vivo for greater than 2 years. In anotherembodiment, T-cells having an altered expression of a BACH2 encodedprotein maintain or increase their proliferation rate in vivo forgreater than 3 years. In another embodiment, T-cells having an alteredexpression of a BACH2 encoded protein maintain or increase theirproliferation rate in vivo for greater than 4 years. In anotherembodiment, T-cells having an altered expression of a BACH2 encodedprotein maintain or increase their proliferation rate in vivo forgreater than 5 years. In another embodiment, T-cells having an alteredexpression of a BACH2 encoded protein maintain or increase theirproliferation rate in vivo for greater than 10 years. In anotherembodiment, T-cells having an altered expression of a BACH2 encodedprotein maintain or increase their proliferation rate in vivo forgreater than 20 years.

In another embodiment, T-cells having an altered form of a STAT5Bencoded protein maintain or increase their proliferation rate in vivofor greater than 1 year. In another embodiment, T-cells having analtered form of a STAT5B encoded protein maintain or increase theirproliferation rate in vivo for greater than 2 years. In anotherembodiment, T-cells having an altered form of a STAT5B encoded proteinmaintain or increase their proliferation rate in vivo for greater than 3years. In another embodiment, T-cells having an altered form of a STAT5Bencoded protein maintain or increase their proliferation rate in vivofor greater than 4 years. In another embodiment, T-cells having analtered form of a STAT5B encoded protein maintain or increase theirproliferation rate in vivo for greater than 5 years. In anotherembodiment, T-cells having an altered form of a STAT5B encoded proteinmaintain or increase their proliferation rate in vivo for greater than10 years. In another embodiment, T-cells having an altered form of aSTAT5B encoded protein maintain or increase their proliferation rate invivo for greater than 20 years.

In another embodiment, T-cells having an altered form of a BACH2 encodedprotein maintain or increase their proliferation rate in vivo forgreater than 1 year. In another embodiment, T-cells having an alteredform of a BACH2 encoded protein maintain or increase their proliferationrate in vivo for greater than 2 years. In another embodiment, T-cellshaving an altered form of a BACH2 encoded protein maintain or increasetheir proliferation rate in vivo for greater than 3 years. In anotherembodiment, T-cells having an altered form of a BACH2 encoded proteinmaintain or increase their proliferation rate in vivo for greater than 4years. In another embodiment, T-cells having an altered form of a BACH2encoded protein maintain or increase their proliferation rate in vivofor greater than 5 years. In another embodiment, T-cells having analtered form of a BACH2 encoded protein maintain or increase theirproliferation rate in vivo for greater than 10 years. In anotherembodiment, T-cells having an altered form of a BACH2 encoded proteinmaintain or increase their proliferation rate in vivo for greater than20 years.

In another embodiment, CAR T-cells having an altered expression of aSTAT5B encoded protein are maintained for an extended time period orhave an increased proliferation rate in vivo. In another embodiment, CART-cells having an altered expression of a BACH2 encoded protein aremaintained for an extended time period or have an increasedproliferation rate in vivo. In another embodiment, CAR T-cells having analtered form of a STAT5B encoded protein are maintained for an extendedtime period or have an increased proliferation rate in vivo. In anotherembodiment, CAR T-cells having an altered form of a BACH2 encodedprotein are maintained for an extended time period or have an increasedproliferation rate in vivo

In another embodiment, TCR T-cells having an altered expression of aSTAT5B encoded protein are maintained for an extended time period orhave an increased proliferation rate in vivo. In another embodiment, TCRT-cells having an altered expression of a BACH2 encoded protein aremaintained for an extended time period or have an increasedproliferation rate in vivo. In another embodiment, TCR T-cells having analtered form of a STAT5B encoded protein are maintained for an extendedtime period or have an increased proliferation rate in vivo. In anotherembodiment, TCR T-cells having an altered form of a BACH2 encodedprotein are maintained for an extended time period or have an increasedproliferation rate in vivo.

In another embodiment, Treg-cells having an altered expression of aSTAT5B encoded protein maintain or increase their proliferation rate invivo. In another embodiment, Treg-cells having an altered expression ofa BACH2 encoded protein maintain or increase their proliferation rate invivo. In another embodiment, Treg-cells having an altered form of aSTAT5B encoded protein maintain or increase their proliferation rate invivo. In another embodiment, Treg-cells having an altered form of aBACH2 encoded protein maintain or increase their proliferation rate invivo.

In one embodiment, methods for maintaining or increasing theproliferation rate of a genetically modified immune cell are disclosedherein, wherein the method comprises the step of administering apoptoticcells or an apoptotic supernatant. In another embodiment, methods forincreasing the efficacy of a genetically modified immune cell aredisclosed herein, wherein the method comprises the step of administeringan additional agent comprising apoptotic cells, an apoptoticsupernatant, a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof. In anotherembodiment, methods for treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating a cancer or a tumor disclosedherein administer a genetically modified immune cell and an additionalagent, wherein said additional agent comprises apoptotic cells, anapoptotic supernatant, a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, a tellurium-based compound, oran immune modulating agent, or any combination thereof.

Chimeric Antigen Receptor-Expressing T-Cells (CAR T-Cells)

In some embodiments, chimeric antigen receptors (CARs) are a type ofantigen-targeted receptor composed of intracellular T-cell signalingdomains fused to extracellular tumor-binding moieties, most commonlysingle-chain variable fragments (scFvs) from monoclonal antibodies. CARsdirectly recognize cell surface antigens, independent of MHC-mediatedpresentation, permitting the use of a single receptor construct specificfor any given antigen in all patients. Initial CARs fusedantigen-recognition domains to the CD3ζ activation chain of the T-cellreceptor (TCR) complex. While these first generation CARs induced T-celleffector function in vitro, they were largely limited by poor antitumorefficacy in vivo. Subsequent CAR iterations have included secondarycostimulatory signals in tandem with CD3ζ, including intracellulardomains from CD28 or a variety of TNF receptor family molecules such as4-1BB (CD137) and OX40 (CD134). Further, third generation receptorsinclude two costimulatory signals in addition to CD3ζ, most commonlyfrom CD28 and 4-1BB. Second and third generation CARs dramaticallyimproved antitumor efficacy, in some cases inducing complete remissionsin patients with advanced cancer.

In some embodiments, a CAR T-cell is an immunoresponsive cell comprisingan antigen receptor, which is activated when its receptor binds to itsantigen.

In some embodiments, the CAR T-cells used in the compositions andmethods as disclosed herein are first generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are second generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are third generation CAR T-cells. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein are fourth generation CAR T-cells. In some embodiments,each generation of CAR T-cells is more potent than the CAR T-cells ofearlier generations.

In some embodiments, first-generation CARs have one signaling domain,typically the cytoplasmic signaling domain of the CD3 TCRζ chain.

In another embodiment, the CAR T-cells as disclosed herein are secondgeneration CAR T-cells. In another embodiment, CAR T-cells as disclosedherein comprise a tripartite chimeric receptor (TPCR). In someembodiments, CAR T-cells as disclosed herein, comprise one or moresignaling moieties that activate naïve T-cells in a co-stimulationindependent manner. In another embodiment, the CAR T-cells furtherencode one or more members of the tumor necrosis factor receptor family,which in some embodiments, is CD27, 4-1BB (CD137), or OX40 (CD134), or acombination thereof.

Third-generation CAR T-cells attempt to harness the signaling potentialof 2 costimulatory domains: in some embodiments, the CD28 domainfollowed by either the 4-1BB or OX-40 signaling domains. In anotherembodiment, the CAR T-cells used in the compositions and methods asdisclosed herein further encode a co-stimulatory signaling domain, whichin one embodiment is CD28. In another embodiment, the signaling domainis the CD3ζ-chain, CD97, GDI 1a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40,4-1BB, CD28 signaling domain, or combinations thereof.

In some embodiments, telomere length and replicative capacity correlatewith the engraftment efficiency and antitumor efficacy of adoptivelytransferred T-cell lines. In some embodiments, CD28 stimulationmaintains telomere length in T-cells.

In some embodiments, CAR-modified T-cell potency may be further enhancedthrough the introduction of additional genes, including those encodingproliferative cytokines (ie, IL-12) or costimulatory ligands (ie,4-1BBL), thus producing “armored” fourth-generation CAR-modifiedT-cells. In some embodiments, “armored CAR T-cells,” are CAR T-cellswhich are protected from the inhibitory tumor microenvironment. Inanother embodiment, the “armored” CAR technology incorporates the localsecretion of soluble signaling proteins to amplify the immune responsewithin the tumor microenvironment with the goal of minimizing systemicside effects. In some embodiments, the signaling protein signal isIL-12, which can stimulate T-cell activation and recruitment. In someembodiments, “armored” CAR technology is especially useful in solidtumor indications, in which microenvironment and potentimmunosuppressive mechanisms have the potential to make theestablishment of a robust anti-tumor response more challenging.

In some embodiments, CAR T-cells are genetically modified to encodemolecules involved in the prevention of apoptosis, the remodeling of thetumor microenvironment, induction of homeostatic proliferation, andchemokine receptors that promote directed T-cell homing.

In another embodiment, CAR T-cell therapy used in the compositions andmethods as disclosed herein is enhanced using the expression of cytokinetransgenes, combination therapy with small molecule inhibitors, ormonoclonal antibodies. In another embodiment, other strategies aimed atimproving CAR T-cell therapy including using dual CARs and chemokinereceptors to more specifically target tumor cells are to be consideredpart of the CAR T-cells and CAR T-cell therapy as disclosed herein.

In some embodiments, the CAR T-cells of the compositions and methods asdisclosed herein comprise a second binding domain that can lead toeither an inhibitory or amplifying signal, in order to increasespecificity of CAR T-cells for cancer cells versus normal cells. Forexample, a CAR T-cell can be engineered such that it would be triggeredin the presence of one target protein, but if a second protein ispresent it would be inhibited. Alternatively, it could also beengineered such that two target proteins would be required for maximalactivation. These approaches may increase the specificity of the CAR fortumor relative to normal tissue.

In some embodiments, the CAR T-cells used in the compositions andmethods as disclosed herein encode antibody-based external receptorstructures and cytosolic domains that encode signal transduction modulescomposed of the immunoreceptor tyrosine-based activation motif.

In some embodiments, the CAR T-cell further encodes a single-chainvariable fragment (scFv) that binds a polypeptide that hasimmunosuppressive activity. In another embodiment, the polypeptide thathas immunosuppressive activity is CD47, PD-1, CTLA-4, or a combinationthereof.

In some embodiments, the CAR T-cell further encodes a single-chainvariable fragment (scFv) that binds a polypeptide that hasimmunostimulatory activity. In another embodiment, the polypeptide thathas immunostimulatory activity is CD28, OX-40, 4-1 BB or a combinationthereof. In another embodiment, the CAR T-cell further encodes a CD40ligand (CD40L), which, in some embodiments, enhances theimmunostimulatory activity of the antigen.

In some embodiments, the immune cells are cytotoxic. In anotherembodiment, cytotoxic cells for genetic modification can be obtainedfrom bone marrow of the subject or a donor. In other cases, the cellsare obtained from a stem cell. For example, cytotoxic cells can bederived from human pluripotent stem cells such as human embryonic stemcells or human induced pluripotent T-cells. In the case of inducedpluripotent stem cells (IPSCs), such pluripotent T-cells can be obtainedusing a somatic cell from the subject to which genetically modifiedcytotoxic cells will be provided. In some embodiments, immune cells maybe obtained from a subject or donor by harvesting cells by venipuncture,by apheresis methods, by white cell mobilization followed by apheresisor venipuncture, or by bone marrow aspiration.

In some embodiments, a method as disclosed herein comprises obtainingimmune cells from a subject, and genetically modifying the immune cellsto express a chimeric antigen receptor. In another embodiment, a methodas disclosed herein comprises obtaining immune cells from a subject,genetically modifying the immune cells to express a chimeric antigenreceptor and combining with apoptotic cell population resulting inreduced cytokine production in a subject but substantially unaffectedcytotoxicity relative to immune cells expressing a CAR not administeredwith an apoptotic cell population (FIGS. 1A-1B and 2 ). In anotherembodiment, a method as disclosed herein comprises obtaining immunecells from a subject, genetically modifying the immune cells to expressa chimeric antigen receptor and combining with an apoptotic cellsupernatant or a composition comprising the supernatant, resulting inreduced cytokine production in a subject but substantially unaffectedcytotoxicity relative to immune cells expressing a CAR not administeredwith an apoptotic cell supernatant. In another embodiment,administration of an apoptotic cell population or a supernatant fromapoptotic cells does not reduce the efficacy of the immune cellsexpressing the chimeric antigen receptor.

In one embodiment, disclosed herein are immune cells, in someembodiments, CAR T-cells in which the T-cell is autologous to thesubject. In another embodiment, the CAR T-cells are heterologous to thesubject. In some embodiments, the CAR T-cells are allogeneic. In someembodiments, the CAR T-cells are universal allogeneic CAR T-cells. Inanother embodiment, the T-cells may be autologous, allogeneic, orderived in vitro from engineered progenitor or stem cells.

In another embodiment, the CAR T-cells and apoptotic cells describedherein, are both derived from the same source. In a further embodiment,the CAR T-cells and apoptotic cells described herein, are both derivedfrom the subject (FIG. 1 ). In an alternative embodiment, the CART-cells and apoptotic cells described herein, are derived from differentsources. In yet another embodiment, the CAR T-cells are autologous andthe apoptotic cells described herein, are allogeneic (FIG. 2 ). Askilled artisan would appreciate that similarly, an apoptotic cellsupernatant may be made from cells derived from the same source as theCAR T-cell, which may in one embodiment be autologous cells, or anapoptotic cell supernatant may be made from cells derived from a sourcedifferent from the source of CAR T-cells.

A skilled artisan would appreciate that the term “heterologous” mayencompass a tissue, cell, nucleic acid molecule or polypeptide that isderived from a different organism. In some embodiments, a heterologousprotein is a protein that was initially cloned from or derived from adifferent T-cell type or a different species from the recipient and thatis not normally present in a cell or sample obtained from a cell.

Accordingly, one embodiment as disclosed herein relates to cytotoxicimmune cells (e.g., NK cells or T-cells) comprising chimeric antigenreceptors (CARs) whereby the cells retain their cytotoxic function. Inanother embodiment, the chimeric antigen receptor is exogenous to theT-cell. In another embodiment, the CAR is recombinantly expressed. Inanother embodiment, the CAR is expressed from a vector.

In some embodiments, the T-cell utilized to generate CAR T-cells is anaïve CD4⁺ T-cell. In another embodiment, the T-cell utilized togenerate CAR T-cells is a naïve CD8⁺ T-cell. In another embodiment, theT-cell utilized to generate CAR T-cells is an effector T-cell. Inanother embodiment, the T-cell utilized to generate CAR T-cells is aregulatory T-cell (Treg). In another embodiment, the T-cell utilized togenerate CAR T-cells is a cytotoxic T-cell.

Sources for genetically modified immune cells, for example T cells, havebeen described extensively in the literature, see for example Themelliet al. (2015) New Cell Sources for T Cell Engineering and AdoptiveImmunotherapy. Cell Stem Cell 16: 357-366; Han et al. (2013) Journal ofHematology & Oncology 6:47-53; Wilkie et al. (2010) J Bio Chem285(33):25538-25544; and van der Stegen et al. (2013) J. Immunol 191:4589-4598. CAR T-cells are available to order from a commercial sourcesuch as Creative Biolabs (NY USA), which provides custom constructionand production services for Chimeric Antigen Receptors (CAR) and alsoprovides premade CAR constructs stock, which can induce protectiveimmunity encode by recombinant adenovirus vaccine. Custom made CART-cells may also be obtained from Promab Biotechnologies (CA USA), whichcan provide specifically designed CAR T-cells.

T-Cell Receptors (TCRs) Cells

In one embodiment, compositions and methods as disclosed herein utilizea designer T-cell receptor (TCR) cells in addition to or in place of CART-cells. The TCR is a multi-subunit transmembrane complex that mediatesthe antigen-specific activation of T-cells. The TCR is composed of twodifferent polypeptide chains. The TCR confers antigenic specificity onthe T cell, by recognizing an antigen epitope on the target cell, forexample a tumor or cancer cell. Following contact with the antigenpresent on the tumor or cancer cell, T-cells proliferate and acquire thephenotype and function to allow them eliminate the cancer or tumorcells.

In one embodiment, TCR T-cell therapy comprises introducing a T-cellreceptor (TCR) that is specific to an epitope of a protein of interestinto a T-cell. In another embodiment, the protein of interest is atumor-associated antigen. In another embodiment, the geneticallyengineered TCR recognizes a tumor antigen epitope presented by the majorhistocompatibility complex (MHC) on the tumor cell along with T-cellactivating domains. In another embodiment, the T-cell receptorsrecognize antigens irrespectively of their intracellular or membranelocalization. In another embodiment, TCRs recognize tumor cells thatintracellularly express a tumor associated antigen. In one embodimentTCRs recognize internal antigens. In another embodiment, TCRs recognizeangiogenic factors. In another embodiment, an angiogenic factor is amolecule involved in the formation of new blood vessels. Variousgenetically modified T-cell receptors and methods of their productionare known in the art.

In one embodiment, TCR T-cell therapy is used to treat, prevent,inhibit, ameliorate, reduce the incidence of, or alleviate a cancer or atumor. In one embodiment, TCR T-cell therapy is used to treat, prevent,inhibit, ameliorate, reduce the incidence of, or alleviate advancedmetastatic disease, including those with hematological (lymphoma andleukemia) and solid tumors (refractory melanoma, sarcoma). In oneembodiment, the TCR T-cell therapy used in the compositions and methodsas disclosed herein treat a malignancy listed in Table 1 of Sadelain etal., (Cancer Discov. 2013 April; 3(4): 388-398).

In another embodiment, the T-cell receptor is genetically modified tobind NY-ESO-1 epitopes, and the TCR-engineered T-cell is anti-NY-ESO-1.In another embodiment, the T-cell receptor is genetically modified tobind HPV-16 E6 epitopes, and the TCR-engineered T-cell is anti-HPV-16E6. In another embodiment, the T-cell receptor is genetically modifiedto bind HPV-16 E7 epitopes, and the TCR-engineered T-cell is anti-HPV-16E7. In another embodiment, the T-cell receptor is genetically modifiedto bind MAGE A3/A6 epitopes, and the TCR-engineered T-cell is anti-MAGEA3/A6. In another embodiment, the T-cell receptor is geneticallymodified to bind MAGE A3 epitopes, and the TCR-engineered T-cell isanti-MAGE A3. In another embodiment, the T-cell receptor is geneticallymodified to bind SSX2 epitopes, and the TCR-engineered T-cell isanti-SSX2. In another embodiment, the T-cell receptor is geneticallymodified to bind a target antigen disclosed herein. Using the tools wellknown in the art, a skilled would appreciate that the T-cell receptormay be genetically modified to bind a target antigen present on a canceror tumor cell, wherein the TCR-engineer T-cell comprises an anti-tumoror anti-cancer cell.

In one embodiment, a method as disclosed herein comprises obtainingimmune cells from a subject, and genetically modifying the immune cellsto express a recombinant T-cell receptor (TCR). In another embodiment, amethod as disclosed herein comprises obtaining immune cells from asubject, genetically modifying the immune cells to express a recombinantTCR and combining with an additional agent, wherein said additionalagent comprises an apoptotic cell population, an apoptotic cellsupernatant, a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof.

In one embodiment, the T-cell utilized to generate TCR T-cells is anaïve CD4⁺ T-cell. In another embodiment, the T-cell utilized togenerate TCR T-cells is a naïve CD8⁺ T-cell. In another embodiment, theT-cell utilized to generate TCR T-cells is an effector T-cell. Inanother embodiment, the T-cell utilized to generate TCR T-cells is aregulatory T-cell (Treg). In another embodiment, the T-cell utilized togenerate TCR T-cells is a cytotoxic T-cell.

TCR T-cells have been described extensively in the literature, see forexample Sharpe and Mount (2015) ibid.; Essand M, Loskog ASI (2013)Genetically engineered T cells for the treatment of cancer (Review). JIntern Med 273: 166-181; and Kershaw et al. (2014) Clinical applicationof genetically modified T cells in cancer therapy. Clinical &Translational Immunology 3:1-7.

Targeting Antigens

In some embodiments, the CAR binds to an epitope of an antigen via anantibody or an antibody fragment that is directed to the antigen. Inanother embodiment, the antibody is a monoclonal antibody. In anotherembodiment, the antibody is a polyclonal antibody. In anotherembodiment, the antibody fragment is a single-chain variable fragment(scFv).

In one embodiment, the TCR binds to an epitope of an antigen via agenetically modified T-cell receptor.

In another embodiment, the CAR T-cells of the compositions as disclosedherein bind to a tumor associated antigen (TAA). In another embodiment,said tumor associated antigen is: Mucin 1, cell surface associated(MUC1) or polymorphic epithelial mucin (PEM), Arginine-rich, mutated inearly stage tumors (Armet), Heat Shock Protein 60 (HSP60), calnexin(CANX), methylenetetrahydrofolate dehydrogenase (NADP+dependent) 2,methenyltetrahydrofolate cyclohydrolase (MTHFD2), fibroblast activationprotein (FAP), matrix metallopeptidase (MMP6), B Melanoma Antigen-1(BAGE-1), aberrant transcript of N-acetyl glucosaminyl transferase V(GnTV), Q5H943, Carcinoembryonic antigen (CEA), PmeI, Kallikrein-4,Mammaglobin-1, MART-1, GPR143-OA1, prostate specific antigen (PSA),TRP1, Tyrosinase, FGP-5, NEU proto-oncogene, Aft, MMP-2, prostatespecific membrane antigen (PSMA), Telomerase-associated protein-2,Prostatic acid phosphatase (PAP), Uroplakin II or Proteinase 3.

In another embodiment, the CAR binds to CD19 or CD20 to target B cellsin the case where one would like to destroy B cells as in leukemia. CD19is a B cell lineage specific surface receptor whose broad expression,from pro-B cells to early plasma cells, makes it an attractive targetfor the immunotherapy of B cell malignancies. In another embodiment, theCAR binds to ROR1, CD22, or GD2. In another embodiment, the CAR binds toNY-ESO-1. In another embodiment, the CAR binds to MAGE family proteins.In another embodiment, the CAR binds to mesothelin. In anotherembodiment, the CAR binds to c-erbB2. In another embodiment, the CARbinds to mutational antigens that are tumor specific, such as BRAFV600Emutations and BCR-ABL translocations. In another embodiment, the CARbinds to viral antigens which are tumor-specific, such as EBV in HD, HPVin cervical cancer, and polyomavirus in Merkel cancer. In anotherembodiment, the CAR T-cell binds to Her2/neu. In another embodiment, theCAR T-cell binds to EGFRvIII.

In some embodiments, the chimeric antigen receptor (CAR) T-cell bindsthe CD19 antigen. In another embodiment, the CAR binds the CD22 antigen.In another embodiment, the CAR binds to alpha folate receptor. Inanother embodiment, the CAR binds to CAIX. In another embodiment, theCAR binds to CD20. In another embodiment, the CAR binds to CD23. Inanother embodiment, the CAR binds to CD24. In another embodiment, theCAR binds to CD30. In another embodiment, the CAR binds to CD33. Inanother embodiment, the CAR binds to CD38. In another embodiment, theCAR binds to CD44v6. In another embodiment, the CAR binds to CD44v7/8.In another embodiment, the CAR binds to CD123. In another embodiment,the CAR binds to CD171. In another embodiment, the CAR binds tocarcinoembryonic antigen (CEA). In another embodiment, the CAR binds toEGFRvIII. In another embodiment, the CAR binds to EGP-2. In anotherembodiment, the CAR binds to EGP-40. In another embodiment, the CARbinds to EphA2. In another embodiment, the CAR binds to Erb-B2. Inanother embodiment, the CAR binds to Erb-B 2,3,4. In another embodiment,the CAR binds to Erb-B3/4. In another embodiment, the CAR binds to FBP.In another embodiment, the CAR binds to fetal acetylcholine receptor. Inanother embodiment, the CAR binds to G_(D2). In another embodiment, theCAR binds to G_(D3). In another embodiment, the CAR binds to HER2. Inanother embodiment, the CAR binds to HMW-MAA. In another embodiment, theCAR binds to IL-11Ralpha. In another embodiment, the CAR binds toIL-13Ralpha1. In another embodiment, the CAR binds to KDR. In anotherembodiment, the CAR binds to kappa-light chain. In another embodiment,the CAR binds to Lewis Y. In another embodiment, the CAR binds to L-celladhesion molecule. In another embodiment, the CAR binds to MAGE-A1. Inanother embodiment, the CAR binds to mesothelin. In another embodiment,the CAR binds to CMV infected cells. In another embodiment, the CARbinds to MUC1. In another embodiment, the CAR binds to MUC16. In anotherembodiment, the CAR binds to NKG2D ligands. In another embodiment, theCAR binds to NY-ESO-1 (amino acids 157-165). In another embodiment, theCAR binds to oncofetal antigen (h5T4). In another embodiment, the CARbinds to PSCA. In another embodiment, the CAR binds to PSMA. In anotherembodiment, the CAR binds to ROR1. In another embodiment, the CAR bindsto TAG-72. In another embodiment, the CAR binds to VEGF-R2 or other VEGFreceptors. In another embodiment, the CAR binds to B7-H6. In anotherembodiment, the CAR binds to CA9. In another embodiment, the CAR bindsto α_(v)β₆ integrin. In another embodiment, the CAR binds to 8H9. Inanother embodiment, the CAR binds to NCAM. In another embodiment, theCAR binds to fetal acetylcholine receptor.

In another embodiment, the chimeric antigen receptor (CAR) T-celltargets the CD19 antigen, and has a therapeutic effect on subjects withB-cell malignancies, ALL, Follicular lymphoma, CLL, and Lymphoma. Inanother embodiment, the CAR T-cell targets the CD22 antigen, and has atherapeutic effect on subjects with B-cell malignancies. In anotherembodiment, the CAR T-cell targets alpha folate receptor or folatereceptor alpha, and has a therapeutic effect on subjects with ovariancancer or epithelial cancer. In another embodiment, the CAR T-celltargets CAIX or G250/CAIX, and has a therapeutic effect on subjects withrenal cell carcinoma. In another embodiment, the CAR T-cell targetsCD20, and has a therapeutic effect on subjects with Lymphomas, B-cellmalignancies, B-cell lymphomas, Mantle cell lymphoma and, indolentB-cell lymphomas. In another embodiment, the CAR T-cell targets CD23,and has a therapeutic effect on subjects with CLL. In anotherembodiment, the CAR T-cell targets CD24, and has a therapeutic effect onsubjects with pancreatic adenocarcinoma. In another embodiment, the CART-cell targets CD30, and has a therapeutic effect on subjects withLymphomas or Hodgkin lymphoma. In another embodiment, the CAR T-celltargets CD33, and has a therapeutic effect on subjects with AML. Inanother embodiment, the CAR T-cell targets CD38, and has a therapeuticeffect on subjects with Non-Hodgkin lymphoma. In another embodiment, theCAR T-cell targets CD44v6, and has a therapeutic effect on subjects withseveral malignancies. In another embodiment, the CAR T-cell targetsCD44v7/8, and has a therapeutic effect on subjects with cervicalcarcinoma. In another embodiment, the CAR T-cell targets CD123, and hasa therapeutic effect on subjects with myeloid malignancies. In anotherembodiment, the CAR T-cell targets CEA, and has a therapeutic effect onsubjects with colorectal cancer. In another embodiment, the CAR T-celltargets EGFRvIII, and has a therapeutic effect on subjects withGlioblastoma. In another embodiment, the CAR T-cell targets EGP-2, andhas a therapeutic effect on subjects with multiple malignancies. Inanother embodiment, the CAR T-cell targets EGP-40, and has a therapeuticeffect on subjects with colorectal cancer. In another embodiment, theCAR T-cell targets EphA2, and has a therapeutic effect on subjects withGlioblastoma. In another embodiment, the CAR T-cell targets Erb-B2 orErbB3/4, and has a therapeutic effect on subjects with Breast cancer andothers, prostate cancer, colon cancer, various tumors. In anotherembodiment, the CAR T-cell targets Erb-B 2,3,4, and has a therapeuticeffect on subjects with Breast cancer and others. In another embodiment,the CAR T-cell targets FBP, and has a therapeutic effect on subjectswith Ovarian cancer. In another embodiment, the CAR T-cell targets fetalacetylcholine receptor, and has a therapeutic effect on subjects withRhabdomyosarcoma. In another embodiment, the CAR T-cell targets GD2, andhas a therapeutic effect on subjects with Neuroblastoma, melanoma, orEwing's sarcoma. In another embodiment, the CAR T-cell targets GD3, andhas a therapeutic effect on subjects with Melanoma. In anotherembodiment, the CAR T-cell targets HER2, and has a therapeutic effect onsubjects with medulloblastoma, pancreatic adenocarcinoma, Glioblastoma,Osteosarcoma, or Ovarian cancer. In another embodiment, the CAR T-celltargets HMW-MAA, and has a therapeutic effect on subjects with Melanoma.In another embodiment, the CAR T-cell targets IL-11Ralpha, and has atherapeutic effect on subjects with Osteosarcoma. In another embodiment,the CAR T-cell targets IL-13Ralpha1, and has a therapeutic effect onsubjects with Glioma, Glioblastoma, or medulloblastoma. In anotherembodiment, the CAR T-cell targets IL-13 receptor alpha2, and has atherapeutic effect on subjects with several malignancies. In anotherembodiment, the CAR T-cell targets KDR, and has a therapeutic effect onsubjects with tumors by targeting tumor neovasculature. In anotherembodiment, the CAR T-cell targets kappa-light chain, and has atherapeutic effect on subjects with B-cell malignancies (B-NHL, CLL). Inanother embodiment, the CAR T-cell targets Lewis Y, and has atherapeutic effect on subjects with various carcinomas orepithelial-derived tumors. In another embodiment, the CAR T-cell targetsL-cell adhesion molecule, and has a therapeutic effect on subjects withNeuroblastoma. In another embodiment, the CAR T-cell targets MAGE-A1 orHLA-A1 MAGE A1, and has a therapeutic effect on subjects with Melanoma.In another embodiment, the CAR T-cell targets mesothelin, and has atherapeutic effect on subjects with Mesothelioma. In another embodiment,the CAR T-cell targets CMV infected cells, and has a therapeutic effecton subjects with CMV. In another embodiment, the CAR T-cell targetsMUC1, and has a therapeutic effect on subjects with breast or ovariancancer. In another embodiment, the CAR T-cell targets MUC16, and has atherapeutic effect on subjects with ovarian cancer. In anotherembodiment, the CAR T-cell targets NKG2D ligands, and has a therapeuticeffect on subjects with myeloma, ovarian, and other tumors. In anotherembodiment, the CAR T-cell targets NY-ESO-1 (157-165) or HLA-A2NY-ESO-1, and has a therapeutic effect on subjects with multiplemyeloma. In another embodiment, the CAR T-cell targets oncofetal antigen(h5T4), and has a therapeutic effect on subjects with various tumors. Inanother embodiment, the CAR T-cell targets PSCA, and has a therapeuticeffect on subjects with prostate carcinoma. In another embodiment, theCAR T-cell targets PSMA, and has a therapeutic effect on subjects withprostate cancer/tumor vasculature. In another embodiment, the CAR T-celltargets ROR1, and has a therapeutic effect on subjects with B-CLL andmantle cell lymphoma. In another embodiment, the CAR T-cell targetsTAG-72, and has a therapeutic effect on subjects with adenocarcinomas orgastrointestinal cancers. In another embodiment, the CAR T-cell targetsVEGF-R2 or other VEGF receptors, and has a therapeutic effect onsubjects with tumors by targeting tumor neovasculature. In anotherembodiment, the CAR T-cell targets CA9, and has a therapeutic effect onsubjects with renal cell carcinoma. In another embodiment, the CART-cell targets CD171, and has a therapeutic effect on subjects withrenal neuroblastoma. In another embodiment, the CAR T-cell targets NCAM,and has a therapeutic effect on subjects with neuroblastoma. In anotherembodiment, the CAR T-cell targets fetal acetylcholine receptor, and hasa therapeutic effect on subjects with rhabdomyosarcoma. In anotherembodiment, the CAR binds to one of the target antigens listed in Table1 of Sadelain et al. (Cancer Discov. 2013 April; 3(4): 388-398), whichis incorporated by reference herein in its entirety. In anotherembodiment, CAR T-cells bind to carbohydrate or glycolipid structures.

In one embodiment the CAR T-cells binds to an angiogenic factor, therebytargeting tumor vasculature. In some embodiments, the angiogenic factoris VEGFR2. in another embodiment, the angiogenic factor is endoglin. Inanother embodiment, an angiogenic factor disclosed herein is Angiogenin;Angiopoietin-1; Del-1; Fibroblast growth factors: acidic (aFGF) andbasic (bFGF); Follistatin; Granulocyte colony-stimulating factor(G-CSF); Hepatocyte growth factor (HGF)/scatter factor (SF);Interleukin-8 (IL-8); Leptin; Midkine; Placental growth factor;Platelet-derived endothelial cell growth factor (PD-ECGF);Platelet-derived growth factor-BB (PDGF-BB); Pleiotrophin (PTN);Progranulin; Proliferin; Transforming growth factor-alpha (TGF-alpha);Transforming growth factor-beta (TGF-beta); Tumor necrosis factor-alpha(TNF-alpha); Vascular endothelial growth factor (VEGF)/vascularpermeability factor (VPF). In another embodiment, an angiogenic factoris an angiogenic protein. In some embodiments, a growth factor is anangiogenic protein. In some embodiments, an angiogenic protein for usein the compositions and methods disclosed herein is Fibroblast growthfactors (FGF); VEGF; VEGFR and Neuropilin 1 (NRP-1); Angiopoietin 1(Ang1) and Tie2; Platelet-derived growth factor (PDGF; BB-homodimer) andPDGFR; Transforming growth factor-beta (TGF-β), endoglin and TGF-βreceptors; monocyte chemotactic protein-1 (MCP-1); Integrins αVβ3, αVβ5and α5β1; VE-cadherin and CD31; ephrin; plasminogen activators;plasminogen activator inhibitor-1; Nitric oxide synthase (NOS) andCOX-2; AC133; or Id1/Id3. In some embodiments, an angiogenic protein foruse in the compositions and methods disclosed herein is an angiopoietin,which in some embodiments, is Angiopoietin 1, Angiopoietin 3,Angiopoietin 4 or Angiopoietin 6. In some embodiments, endoglin is alsoknown as CD105; EDG; HHT1; ORW; or ORW1. In some embodiments, endoglinis a TGFbeta co-receptor.

In another embodiment, the CAR T-cells bind to an antigen associatedwith an infectious agent. In some embodiments, the infectious agent isMycobacterium tuberculosis. In some embodiments, said Mycobacteriumtuberculosis associated antigen is: Antigen 85B, Lipoprotein IpqH, ATPdependent helicase putative, uncharacterized protein Rv0476/MTO4941precursor or uncharacterized protein Rv1334/MT1376 precursor.

In another embodiment, the CAR T-cells binds to an antibody. In someembodiments, the CAR T-cell is an “antibody-coupled T-cell receptor”(ACTR). According to this embodiment, the CAR T-cell is a universal CART-cell. In another embodiment, the CAR T-cell having an antibodyreceptor is administered before, after, or at the same time as theantibody is administered and then binds to the antibody, bringing theT-cell in close proximity to the tumor or cancer. In another embodiment,the antibody is directed against a tumor cell antigen. In anotherembodiment, the antibody is directed against CD20. In anotherembodiment, the antibody is rituximab.

In another embodiment, the antibody is Trastuzumab (Herceptin;Genentech): humanized IgG1, which is directed against ERBB2. In anotherembodiment, the antibody is Bevacizumab (Avastin; Genentech/Roche):humanized IgG1, which is directed against VEGF. In another embodiment,the antibody is Cetuximab (Erbitux; Bristol-Myers Squibb): chimerichuman-murine IgG1, which is directed against EGFR. In anotherembodiment, the antibody is Panitumumab (Vectibix; Amgen): human IgG2,which is directed against EGFR. In another embodiment, the antibody isIpilimumab (Yervoy; Bristol-Myers Squibb): IgG1, which is directedagainst CTLA4.

In another embodiment, the antibody is Alemtuzumab (Campath; Genzyme):humanized IgG1, which is directed against CD52. In another embodiment,the antibody is Ofatumumab (Arzerra; Genmab): human IgG1, which isdirected against CD20. In another embodiment, the antibody is Gemtuzumabozogamicin (Mylotarg; Wyeth): humanized IgG4, which is directed againstCD33. In another embodiment, the antibody is Brentuximab vedotin(Adcetris; Seattle Genetics): chimeric IgG1, which is directed againstCD30. In another embodiment, the antibody is 90Y-labelled ibritumomabtiuxetan (Zevalin; IDEC Pharmaceuticals): murine IgG1, which is directedagainst CD20. In another embodiment, the antibody is 131I-labelledtositumomab (Bexxar; GlaxoSmithKline): murine IgG2, which is directedagainst CD20.

In another embodiment, the antibody is Ramucirumab, which is directedagainst vascular endothelial growth factor receptor-2 (VEGFR-2). Inanother embodiment, the antibody is ramucirumab (Cyramza Injection, EliLilly and Company), blinatumomab (BLINCYTO, Amgen Inc.), pembrolizumab(KEYTRUDA, Merck Sharp & Dohme Corp.), obinutuzumab (GAZYVA, Genentech,Inc.; previously known as GA101), pertuzumab injection (PERJETA,Genentech, Inc.), or denosumab (Xgeva, Amgen Inc.). In anotherembodiment, the antibody is Basiliximab (Simulect; Novartis). In anotherembodiment, the antibody is Daclizumab (Zenapax; Roche).

In another embodiment, the antibody to which the CAR T-cell is coupledis directed to a tumor or cancer antigen or a fragment thereof, that isdescribed herein and/or that is known in the art. In another embodiment,the antibody to which the CAR T-cell is couples is directed to atumor-associated antigen. In another embodiment, the antibody to whichthe CAR T-cell is couples is directed to a tumor-associated antigen or afragment thereof that is an angiogenic factor.

In another embodiment, the antibody to which the CAR T-cell is coupledis directed to a tumor or cancer antigen or a fragment thereof, that isdescribed herein and/or that is known in the art.

In some embodiments, antibodies described herein may be used incombination with compositions described herein, for example but notlimited to a composition comprising CAR-T cells or early apoptoticcells, or any combination thereof.

Cytokine Storm and Cytokine Release Syndrome

In one embodiment, a method as disclosed herein includes providingimmune cells, such as NK cells, dendritic cells, TCR T-cells, or T-cellscomprising engineered chimeric antigen receptors (CAR T-cells), with atleast an additional agent to decrease toxic cytokine release or“cytokine release syndrome” (CRS) or “severe cytokine release syndrome”(sCRS) or “cytokine storm” that may occur in the subject. In anotherembodiment the CRS, sCRS or cytokine storm occurs as a result ofadministration of the immune cells. In another embodiment, the CRS, sCRSor cytokine storm is the result of a stimulus, condition, or syndromeseparate from the immune cells (see below). In another embodiment, acytokine storm, cytokine cascade, or hypercytokinemia is a more severeform of cytokine release syndrome.

In one embodiment, the additional agent for decreasing harmful cytokinerelease comprises apoptotic cells or a composition comprising saidapoptotic cells. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises an apoptotic cellsupernatant or a composition comprising said supernatant. In anotherembodiment, the additional agent for decreasing harmful cytokine releasecomprises a CTLA-4 blocking agent. In another embodiment, the additionalagent for decreasing harmful cytokine release comprises apoptotic cellsor apoptotic cell supernatants or compositions thereof, and a CTLA-4blocking agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises an alpha-1 anti-trypsin orfragment thereof or analogue thereof. In another embodiment, theadditional agent for decreasing harmful cytokine release comprisesapoptotic cells or apoptotic cell supernatants or compositions thereof,and an alpha-1 anti-trypsin or fragment thereof or analogue thereof. Inanother embodiment, the additional agent for decreasing harmful cytokinerelease comprises a tellurium-based compound. In another embodiment, theadditional agent for decreasing harmful cytokine release comprisesapoptotic cells or apoptotic cell supernatants or compositions thereof,and a tellurium-based compound. In another embodiment, the additionalagent for decreasing harmful cytokine release comprises an immunemodulating agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises apoptotic cells orapoptotic cell supernatants or compositions thereof, and an immunemodulating agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises Treg cells. In anotherembodiment, the additional agent for decreasing harmful cytokine releasecomprises apoptotic cells or apoptotic cell supernatants or compositionsthereof, and Treg cells.

A skilled artisan would appreciate that decreasing toxic cytokinerelease or toxic cytokine levels comprises decreasing or inhibitingproduction of toxic cytokine levels in a subject, or inhibiting orreducing the incidence of cytokine release syndrome or a cytokine stormin a subject. In another embodiment toxic cytokine levels are reducedduring CRS or a cytokine storm. In another embodiment, decreasing orinhibiting the production of toxic cytokine levels comprises treatingCRS or a cytokine storm. In another embodiment, decreasing or inhibitingthe production of toxic cytokine levels comprises preventing CRS or acytokine storm. In another embodiment, decreasing or inhibiting theproduction of toxic cytokine levels comprises alleviating CRS or acytokine storm. In another embodiment, decreasing or inhibiting theproduction of toxic cytokine levels comprises ameliorating CRS or acytokine storm. In another embodiment, the toxic cytokines comprisepro-inflammatory cytokines. In another embodiment, pro-inflammatorycytokines comprise IL-6. In another embodiment, pro-inflammatorycytokines comprise IL-1β. In another embodiment, pro-inflammatorycytokines comprise TNF-α, In another embodiment, pro-inflammatorycytokines comprise IL-6, IL-1β, or TNF-α, or any combination thereof.

In one embodiment, cytokine release syndrome is characterized byelevated levels of several inflammatory cytokines and adverse physicalreactions in a subject such as low blood pressure, high fever andshivering. In another embodiment, inflammatory cytokines comprise IL-6,IL-1β, and TNF-α. In another embodiment, CRS is characterized byelevated levels of IL-6, IL-1β, or TNF-α, or any combination thereof. Inanother embodiment, CRS is characterized by elevated levels of IL-8, orIL-13, or any combination thereof. In another embodiment, a cytokinestorm is characterized by increases in TNF-alpha, IFN-gamma, IL-1beta,IL-2, IL-6, IL-8, IL-10, IL-13, GM-CSF, IL-5, fracktalkine, or acombination thereof or a subset thereof. In yet another embodiment, IL-6comprises a marker of CRS or cytokine storm.

In another embodiment, cytokines increased in CRS or a cytokine storm inhumans and mice may comprise any combination of cytokines listed inTables 1 and 2 below.

TABLE 1 Panel of Cytokines Increased in CRS or Cytokine Storm in Humansand/or Mice Human model Mouse model (pre-clinical) Cytokine (clinicalCAR-T (H) Mouse Not Cells secreting this Notes/ (Analyte) trials) originorigin specified cytokine other Flt-3L * DC (?) Fractalkine * APC,Endothelial cells (?) =CX3CL1, Neurotactin (Mouse) M-CSF =CSF1GM-CSF * * (in vitro) T cell, MØ IFN-□ * T cell, MØ, Monocyte IFN-□ ? ?T cell, MØ, Monocyte IFN-□ * * * (in vitro) cytotoxic T cells, helper Tcells, NK cells, MØ, Monocyte, DC IL- 1 □ * Monocyte, MØ, Epithel IL- 1□ * * Macrophages, DCs, fibroblasts, endothelial cells, hepatocytes IL-1 R□ * IL- 2 * * * (in vitro) T cells IL- 2R□ * lymphocytes IL- 4 * * *(in vitro) Th2 cells IL- 5 * * * T cells IL- 6 * * *monocytes/macrophages, dendritic cells, T cells, fibroblasts,keratinocytes, endothelial cells, adipocytes, myocytes, mesangial cells,and osteoblasts IL- 7 * * In vitro by BM stromal cells IL- 8 *Macrophages, monocytes IL - 9 * * T cells, T helper IL- 10 * * * * (invitro) monocytes/macrophages, mast cells, B cells, regulatory T cells,and helper T cells IL- 12 * * MØ, Monocyte, DC, =p70 activatedlymphocytes, (p40 + p35) neutrophils IL- 13 * * T cells

In some embodiments, cytokines Flt-3L, Fractalkine, GM-CSF, IFN-γ,IL-1β, IL-2, IL-2Ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12,and IL-13 of Table 1 are considered to be significant in CRS or cytokinestorm. In another embodiment, IFN-α, IFN-β, IL-1, and IL-1Rα of Table 1appear to be important in CRS or cytokine storm. In another embodiment,M-CSF has unknown importance. In another embodiment, any cytokine listedin Table 1, or combination thereof, may be used as a marker of CRS orcytokine storm.

TABLE 2 Panel of Cytokines Increased in CRS or Cytokine Storm in Humansand/or Mice Human model Mouse model (pre-clinical) Cytokine (clinicalCAR-T (H) Mouse Not Cells secreting this Notes/ (Analyte) trials) originorigin specified cytokine other IL- 15 * * Fibroblasts, monocytes (?) 22IL- 17 * * T cells IL- 18 Macrophages IL- 21 * T helper cells, NK cellsIL- 22 * activated DC and T cells IL- 23 IL- 25 Protective? IL- 27 * APCIP-10 * Monocytes (?) MCP-1 * Endothel, fibroblast, =CXCL10 epithel,monocytes MCP-3 * PBMCs, MØ (?) =CCL2 MIP-1α * * (in vitro) T cells=CXCL9 MIP-1β * T cells =CCL3 PAF ? platelets, endothelial cells, =CCL4neutrophils, monocytes, and macrophages, mesangial cells PGE2 * *Gastrointestinal mucosa and other RANTES * Monocytes TGF-β * * MØ,lymphocytes, =CCL5 endothel, platelets . . . TNF-α * * * * (in vitro)Macrophages, NK cells, T cells TNF-αR1 * HGF MIG * T cellchemoattractant, induced by IFN-γ

In one embodiment, IL-15, IL-17, IL-18, IL-21, IL-22, IP-10, MCP-1,MIP-1α, MIP-1β, and TNF-α of Table 2 are considered to be significant inCRS or cytokine storm. In another embodiment, IL-27, MCP-3, PGE2,RANTES, TGF-β, TNF-αR1, and MIG of Table 2 appear to be important in CRSor cytokine storm. In another embodiment, IL-23 and IL-25 have unknownimportance. In another embodiment, any cytokine listed in Table 2, orcombination thereof, may be used as a marker of CRS or cytokine storm.In another embodiment, mouse cytokines IL-10, IL-1β, IL-2, IP-10, IL-4,IL-5, IL-6, IFNα, IL-9, IL-13, IFN-γ, IL-12p70, GM-CSF, TNF-α, MIP-1α,MIP-1β, IL-17A, IL-15/IL-15R and IL-7 appear to be important in CRS orcytokine storm.

A skilled artisan would appreciate that the term “cytokine” mayencompass cytokines (e.g., interferon gamma (IFN-γ), granulocytemacrophage colony stimulating factor, tumor necrosis factor alpha),chemokines (e.g., MIP 1 alpha, MIP 1 beta, RANTES), and other solublemediators of inflammation, such as reactive oxygen species and nitricoxide.

In one embodiment, increased release of a particular cytokine, whethersignificant, important or having unknown importance, does not a priorimean that the particular cytokine is part of a cytokine storm. In oneembodiment, an increase of at least one cytokine is not the result of acytokine storm or CRS. In another embodiment, CAR T-cells may be thesource of increased levels of a particular cytokine or group ofcytokines.

In another embodiment, cytokine release syndrome is characterized by anyor all of the following symptoms: Fever with or without rigors, malaise,fatigue, anorexia, myalgias, arthalgias, nausea, vomiting, headache SkinRash, Nausea, vomiting, diarrhea, Tachypnea, hypoxemia CardiovascularTachycardia, widened pulse pressure, hypotension, increased cardiacoutput (early), potentially diminished cardiac output (late), ElevatedD-dimer, hypofibrinogenemia with or without bleeding, Azotemia HepaticTransaminitis, hyperbilirubinemia, Headache, mental status changes,confusion, delirium, word finding difficulty or frank aphasia,hallucinations, tremor, dymetria, altered gait, seizures. In anotherembodiment, a cytokine storm is characterized by IL-2 release andlymphoproliferation. In another embodiment, a cytokine storm ischaracterized by increases in cytokines released by CAR T-cells. Inanother embodiment, a cytokine storm is characterized by increases incytokines released by cells other than CAR T-cells.

In another embodiment, cytokine storm leads to potentiallylife-threatening complications including cardiac dysfunction, adultrespiratory distress syndrome, neurologic toxicity, renal and/or hepaticfailure, and disseminated intravascular coagulation.

A skilled artisan would appreciate that the characteristics of acytokine release syndrome (CRS) or cytokine storm are estimated to occura few days to several weeks following the trigger for the CRS orcytokine storm. In one embodiment, CAR T-cells are a trigger for CRS ora cytokine storm. In another embodiment, a trigger for CRS or a cytokinestorm is not CAR T-cells.

In one embodiment, measurement of cytokine levels or concentration, asan indicator of cytokine storm, may be expressed as -fold increase,percent (%) increase, net increase or rate of change in cytokine levelsor concentration. In another embodiment, absolute cytokine levels orconcentrations above a certain level or concentration may be anindication of a subject undergoing or about to experience a cytokinestorm. In another embodiment, absolute cytokine levels or concentrationat a certain level or concentration, for example a level orconcentration normally found in a control subject not undergoing CAR-Tcell therapy, may be an indication of a method for inhibiting orreducing the incidence of a cytokine storm in a subject undergoing CART-cell.

A skilled artisan would appreciate that the term “cytokine level” mayencompass a measure of concentration, a measure of fold change, ameasure of percent (%) change, or a measure of rate change. Further, themethods for measuring cytokines in blood, saliva, serum, urine, andplasma are well known in the art.

In one embodiment, despite the recognition that cytokine storm isassociated with elevation of several inflammatory cytokines, IL-6 levelsmay be used as a common measure of cytokine storm and/or as a commonmeasure of the effectiveness of a treatment for cytokine storms. Askilled artisan would appreciate that other cytokines may be used asmarkers of a cytokine storm, for example TNF-α, IB-1α, IL-8, IL-13, orINF-γ. Further, that assay methods for measuring cytokines are wellknown in the art. A skilled artisan would appreciate that methodsaffecting a cytokine storm may similarly affect cytokine releasesyndrome.

In one embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or a cytokine storm. In another embodiment, disclosedherein is a method of decreasing or inhibiting cytokine production in asubject vulnerable to experiencing cytokine release syndrome or acytokine storm. In another embodiment, methods disclosed herein decreaseor inhibit cytokine production in a subject experiencing cytokinerelease syndrome or a cytokine storm, wherein production of any cytokineor group of cytokines listed in Tables 1 and/or 2 is decreased orinhibited. In another embodiment, cytokine IL-6 production is decreasedor inhibited. In another embodiment, cytokine IL-beta1 production isdecreased or inhibited. In another embodiment, cytokine IL-8 productionis decreased or inhibited. In another embodiment, cytokine IL-13production is decreased or inhibited. In another embodiment, cytokineTNF-alpha production is decreased or inhibited. In another embodiment,cytokines IL-6 production, IL-1beta production, or TNF-alpha production,or any combination thereof is decreased or inhibited.

In one embodiment, cytokine release syndrome is graded. In anotherembodiment, Grade 1 describes cytokine release syndrome in whichsymptoms are not life threatening and require symptomatic treatmentonly, e.g., fever, nausea, fatigue, headache, myalgias, malaise. Inanother embodiment, Grade 2 symptoms require and respond to moderateintervention, such as oxygen, fluids or vasopressor for hypotension. Inanother embodiment, Grade 3 symptoms require and respond to aggressiveintervention. In another embodiment, Grade 4 symptoms arelife-threatening symptoms and require ventilator and patients displayorgan toxicity.

In another embodiment, a cytokine storm is characterized by IL-6 andinterferon gamma release. In another embodiment, a cytokine storm ischaracterized by release of any cytokine or combination thereof, listedin Tables 1 and 2. In another embodiment, a cytokine storm ischaracterized by release of any cytokine or combination thereof, knownin the art.

In one embodiment, symptoms onset begins minutes to hours after theinfusion begins. In another embodiment, symptoms coincide with peakcytokine levels.

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy comprises administering anapoptotic cell population or an apoptotic cell supernatant orcompositions thereof. In another embodiment, the apoptotic cellpopulation or an apoptotic cell supernatant or compositions thereof mayaid the CAR T-cell therapy. In another embodiment, the apoptotic cellpopulation or an apoptotic cell supernatant or compositions thereof mayaid in the inhibition or reducing the incidence of the CRS or cytokinestorm. In another embodiment, the apoptotic cell population or anapoptotic cell supernatant or compositions thereof may aid in treatingthe CRS or cytokine storm. In another embodiment, the apoptotic cellpopulation or an apoptotic cell supernatant or compositions thereof mayaid in preventing the CRS or cytokine storm. In another embodiment, theapoptotic cell population or an apoptotic cell supernatant orcompositions thereof may aid in ameliorating the CRS or cytokine storm.In another embodiment, the apoptotic cell population or an apoptoticcell supernatant or compositions thereof may aid in alleviating the CRSor cytokine storm.

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy, and being administered anapoptotic cell population or an apoptotic cell supernatant orcompositions thereof, comprises administering an additional agent. Inanother embodiment, the additional agent may aid the CAR T-cell therapy.In another embodiment, the additional agent may aid in the inhibition orreducing the incidence of the CRS or cytokine storm. In anotherembodiment, the additional agent may aid in treating the CRS or cytokinestorm. In another embodiment, the additional agent may aid in preventingthe CRS or cytokine storm. In another embodiment, the additional agentmay aid in ameliorating the CRS or cytokine storm. In anotherembodiment, the additional agent may aid in alleviating the CRS orcytokine storm.

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy comprises administering anadditional agent. In another embodiment, the additional agent may aidthe CAR T-cell therapy. In one embodiment, a method of inhibiting orreducing the incidence of a cytokine release syndrome (CRS) or acytokine storm in a subject undergoing TCR T-cell cancer therapycomprises administering an additional agent. In another embodiment, theadditional agent may aid the TCR T-cell therapy. In one embodiment, amethod of inhibiting or reducing the incidence of a cytokine releasesyndrome (CRS) or a cytokine storm in a subject undergoing comprisesadministering an additional agent. In another embodiment, the additionalagent may aid the. In one embodiment, a method of inhibiting or reducingthe incidence of a cytokine release syndrome (CRS) or a cytokine stormin a subject undergoing NK cell therapy comprises administering anadditional agent. In another embodiment, the additional agent may aidthe NK cell therapy.

In another embodiment, the additional agent may aid in the inhibition orreducing the incidence of the CRS or cytokine storm. In anotherembodiment, the additional agent may aid in treating the CRS or cytokinestorm. In another embodiment, the additional agent may aid in preventingthe CRS or cytokine storm. In another embodiment, the additional agentmay aid in ameliorating the CRS or cytokine storm. In anotherembodiment, the additional agent may aid in alleviating the CRS orcytokine storm.

In one embodiment, the additional agent for decreasing harmful cytokinerelease comprises apoptotic cells or a composition comprising saidapoptotic cells. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises an apoptotic cellsupernatant or a composition comprising said supernatant. In anotherembodiment, the additional agent for decreasing harmful cytokine releasecomprises a CTLA-4 blocking agent. In another embodiment, the additionalagent for decreasing harmful cytokine release comprises apoptotic cellsor apoptotic cell supernatants or compositions thereof, and a CTLA-4blocking agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises an alpha-1 anti-trypsin orfragment thereof or analogue thereof. In another embodiment, theadditional agent for decreasing harmful cytokine release comprisesapoptotic cells or apoptotic cell supernatants or compositions thereof,and an alpha-1 anti-trypsin or fragment thereof or analogue thereof. Inanother embodiment, the additional agent for decreasing harmful cytokinerelease comprises a tellurium-based compound. In another embodiment, theadditional agent for decreasing harmful cytokine release comprisesapoptotic cells or apoptotic cell supernatants or compositions thereof,and a tellurium-based compound. In another embodiment, the additionalagent for decreasing harmful cytokine release comprises an immunemodulating agent. In another embodiment, the additional agent fordecreasing harmful cytokine release comprises apoptotic cells orapoptotic cell supernatants or compositions thereof, and an immunemodulating agent.

In another embodiment, compositions and methods as disclosed hereinutilize combination therapy of CAR T-cells with one or moreCTLA-4-blocking agents such as Ipilimumab. In another embodiment,compositions and methods as disclosed herein utilize combined therapycomprising apoptotic cells, CAR T-cells, and one or more CTLA-4-blockingagents. In another embodiment, compositions and methods as disclosedherein utilize combination therapy of TCR T-cells with one or moreCTLA-4-blocking agents such as Ipilimumab. In another embodiment,compositions and methods as disclosed herein utilize combined therapycomprising apoptotic cells, TCR T-cells, and one or more CTLA-4-blockingagents. In another embodiment, compositions and methods as disclosedherein utilize combination therapy of dendritic cells with one or moreCTLA-4-blocking agents such as Ipilimumab. In another embodiment,compositions and methods as disclosed herein utilize combined therapycomprising apoptotic cells, dendritic cells, and one or moreCTLA-4-blocking agents. In another embodiment, compositions and methodsas disclosed herein utilize combination therapy of NK cells with one ormore CTLA-4-blocking agents such as Ipilimumab. In another embodiment,compositions and methods as disclosed herein utilize combined therapycomprising apoptotic cells, NK cells, and one or more CTLA-4-blockingagents.

In another embodiment, CTLA-4 is a potent inhibitor of T-cell activationthat helps to maintain self-tolerance. In another embodiment,administration of an anti-CTLA-4 blocking agent, which in anotherembodiment, is an antibody, produces a net effect of T-cell activation.

In another embodiment, other toxicities resulting from CAR T-cell, TCRT-cell, dendritic cell, or NK cell administration that may be treated,prevented, inhibited, ameliorated, reduced in incidence or alleviated bythe compositions and methods as disclosed herein comprise B cell aplasiaor tumor lysis syndrome (TLS).

In one embodiment, a method of inhibiting or reducing the incidence of acytokine release syndrome (CRS) or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy does not affect the efficacy of theCAR T-cell therapy. In another embodiment, a method of inhibiting orreducing the incidence of CRS or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy, does reduce the efficacy of theCAR T-cells therapy by more than about 5%. In another embodiment, amethod of inhibiting or reducing the incidence of CRS or a cytokinestorm in a subject undergoing CAR T-cell cancer therapy, does reduce theefficacy of the CAR T-cells therapy by more than about 10%. In anotherembodiment, a method of inhibiting or reducing the incidence of CRS or acytokine storm in a subject undergoing CAR T-cell cancer therapy, doesreduce the efficacy of the CAR T-cells therapy by more than about 15%.In another embodiment, a method of inhibiting or reducing the incidenceof CRS or a cytokine storm in a subject undergoing CAR T-cell cancertherapy, does reduce the efficacy of the CAR T-cells therapy by morethan about 20%. In another embodiment, a method of inhibiting orreducing the incidence of CRS or a cytokine storm in a subjectundergoing CAR T-cell cancer therapy, increases the efficacy of the CART-cells therapy by more than about 5%. In another embodiment, a methodof inhibiting or reducing the incidence of CRS or a cytokine storm in asubject undergoing CAR T-cell cancer therapy, increases the efficacy ofthe CAR T-cells therapy by more than about 10%. In another embodiment, amethod of inhibiting or reducing the incidence of CRS or a cytokinestorm in a subject undergoing CAR T-cell cancer therapy, increases theefficacy of the CAR T-cells therapy by more than about 15%. In anotherembodiment, a method of inhibiting or reducing the incidence of CRS or acytokine storm in a subject undergoing CAR T-cell cancer therapy,increases the efficacy of the CAR T-cells therapy by more than about20%.

Any appropriate method of quantifying cytotoxicity can be used todetermine whether activity in an immune cell modified to express a CARremains substantially unchanged. For example, cytotoxicity can bequantified using a cell culture-based assay such as the cytotoxic assaysdescribed in the Examples. Cytotoxicity assays can employ dyes thatpreferentially stain the DNA of dead cells. In other cases, fluorescentand luminescent assays that measure the relative number of live and deadcells in a cell population can be used. For such assays, proteaseactivities serve as markers for cell viability and cell toxicity, and alabeled cell permeable peptide generates fluorescent signals that areproportional to the number of viable cells in the sample. For example acytotoxicity assay may use 7-AAD in a flow cytometry analysis. Kits forvarious cytotoxicity assays are commercially available frommanufacturers such as Promega, Abcam, and Life Technologies.

In another embodiment, a measure of cytotoxicity may be qualitative. Inanother embodiment, a measure of cytotoxicity may be quantitative. In afurther embodiment a measure of cytotoxicity may be related to thechange in expression of a cytotoxic cytokine. In another embodiment, ameasure of cytotoxicity may be determined by survival curve and tumorload in bone marrow and liver.

In one embodiment, the methods as disclosed herein comprise anadditional step that is useful in overcoming rejection of allogeneicdonor cells. In one embodiment, the methods comprise the step of full orpartial lymphodepletion prior to administration of the CAR T-cells,which in one embodiment, are allogeneic CAR T-cells. In anotherembodiment, the lymphodepletion is adjusted so that it delays the hostversus graft reaction for a period sufficient to allow said allogeneicT-cells to attack the tumor to which they are directed, but to an extentinsufficient to require rescue of the host immune system by bone marrowtransplantation. In another embodiment, agents that delay egression ofthe allogeneic T-cells from lymph nodes, such as2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol (FTY720),5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)pheny-1]1,2,4-oxadiazole(SEW2871), 3-(2-(-hexylphenylamino)-2-oxoethylamino)propanoic acid(W123),2-ammonio-4-(2-chloro-4-(3-phenoxyphenylthio)phenyl)-2-(hydroxymethyl)but-ylhydrogen phosphate (KRP-203 phosphate) or other agents known in the art,may be used as part of the compositions and methods as disclosed hereinto allow the use of allogeneic CAR T-cells having efficacy and lackinginitiation of graft vs host disease. In one embodiment, MHC expressionby the allogeneic T-cells is silenced to reduce the rejection of theallogeneic cells. In another embodiment, the apoptotic cells preventrejection of the allogeneic cells.

Cytokine Release Associated with CAR T-Cell Therapy

In one embodiment, cytokine release occurs between a few days to 2 weeksafter administration of immune therapy such as CAR T-cell therapy. Inone embodiment, hypotension and other symptoms follow the cytokinerelease, i.e. from few days to few weeks. Therefore, in one embodiment,apoptotic cells or the apoptotic cell supernatant are administered tosubjects at the same time as immune therapy as prophylaxis. In anotherembodiment, apoptotic cells or supernatant are administered to subjects2-3 days after administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 7 days afteradministration of immune therapy. In another embodiment, apoptotic cellsor supernatant are administered to subjects 10 days after administrationof immune therapy. In another embodiment, apoptotic cells or supernatantare administered to subjects 14 days after administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 2-14 days after administration of immunetherapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant areadministered to subjects 2-3 hours after administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 7 hours after administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 10 hours after administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects14 hours after administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 2-14 hoursafter administration of immune therapy.

In an alternative embodiment, apoptotic cells or the apoptotic cellsupernatant are administered to subjects prior to immune therapy asprophylaxis. In another embodiment, apoptotic cells or supernatant areadministered to subjects 1 day before administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 2-3 days before administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects7 days before administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 10 daysbefore administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 14 daysbefore administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 2-14 daysbefore administration of immune therapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant areadministered to subjects 2-3 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 7 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 10 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 14 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 2-14 hours before administration of immunetherapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant maybe administered therapeutically, once cytokine release syndrome hasoccurred. In one embodiment, apoptotic cells or supernatant may beadministered once cytokine release leading up to or attesting to thebeginning of cytokine release syndrome is detected. In one embodiment,apoptotic cells or supernatant can terminate the increased cytokinelevels, or the cytokine release syndrome, and avoid its sequelae.

In another embodiment, apoptotic cells or apoptotic cell supernatant maybe administered therapeutically, at multiple time points. In anotherembodiment, administration of apoptotic cells or apoptotic cellsupernatant is at least at two time points described herein. In anotherembodiment, administration of apoptotic cells or apoptotic cellsupernatant is at least at three time points described herein. Inanother embodiment, administration of apoptotic cells or apoptotic cellsupernatant is prior to CRS or a cytokine storm, and once cytokinerelease syndrome has occurred, and any combination thereof.

In one embodiment, the chimeric antigen receptor-expressing T-cell (CART-cell) therapy and the apoptotic cell therapy or supernatant areadministered together. In another embodiment, the CAR T-cell therapy isadministered after the apoptotic cell therapy or supernatant. In anotherembodiment, the CAR T-cell therapy is administered prior to theapoptotic cell therapy or supernatant. According to this aspect and inone embodiment, apoptotic cell therapy or supernatant is administeredapproximately 2-3 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy or supernatant is administeredapproximately 6-7 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy or supernatant is administeredapproximately 9 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy is administered up to several monthsafter CAR T-cell therapy.

Therefore, in one embodiment, apoptotic cells or the apoptotic cellsupernatant are administered to subjects at the same time as immunetherapy as prophylaxis. In another embodiment, apoptotic cells orsupernatant are administered to subjects 2-3 days after administrationof immune therapy. In another embodiment, apoptotic cells or supernatantare administered to subjects 7 days after administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 10 days after administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 14 days after administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects2-14 days after administration of immune therapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant areadministered to subjects 2-3 hours after administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 7 hours after administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 10 hours after administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects14 hours after administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 2-14 hoursafter administration of immune therapy.

In an alternative embodiment, apoptotic cells or the apoptotic cellsupernatant are administered to subjects prior to immune therapy asprophylaxis. In another embodiment, apoptotic cells or supernatant areadministered to subjects 1 day before administration of immune therapy.In another embodiment, apoptotic cells or supernatant are administeredto subjects 2-3 days before administration of immune therapy. In anotherembodiment, apoptotic cells or supernatant are administered to subjects7 days before administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 10 daysbefore administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 14 daysbefore administration of immune therapy. In another embodiment,apoptotic cells or supernatant are administered to subjects 2-14 daysbefore administration of immune therapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant areadministered to subjects 2-3 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 7 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 10 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 14 hours before administration of immunetherapy. In another embodiment, apoptotic cells or supernatant areadministered to subjects 2-14 hours before administration of immunetherapy.

In another embodiment, apoptotic cells or apoptotic cell supernatant maybe administered therapeutically, once cytokine release syndrome hasoccurred. In one embodiment, apoptotic cells or supernatant may beadministered once cytokine release leading up to or attesting to thebeginning of cytokine release syndrome is detected. In one embodiment,apoptotic cells or supernatant can terminate the increased cytokinelevels, or the cytokine release syndrome, and avoid its sequelae.

In another embodiment, apoptotic cells or apoptotic cell supernatant maybe administered therapeutically, at multiple time points. In anotherembodiment, administration of apoptotic cells or apoptotic cellsupernatant is at least at two time points described herein. In anotherembodiment, administration of apoptotic cells or apoptotic cellsupernatant is at least at three time points described herein. Inanother embodiment, administration of apoptotic cells or apoptotic cellsupernatant is prior to CRS or a cytokine storm, and once cytokinerelease syndrome has occurred, and any combination thereof.

In one embodiment, the chimeric antigen receptor-expressing T-cell (CART-cell) therapy and the apoptotic cell therapy or supernatant areadministered together. In another embodiment, the CAR T-cell therapy isadministered after the apoptotic cell therapy or supernatant. In anotherembodiment, the CAR T-cell therapy is administered prior to theapoptotic cell therapy or supernatant. According to this aspect and inone embodiment, apoptotic cell therapy or supernatant is administeredapproximately 2-3 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy or supernatant is administeredapproximately 6-7 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy or supernatant is administeredapproximately 9 weeks after the CAR T-cell therapy. In anotherembodiment, apoptotic cell therapy is administered up to several monthsafter CAR T-cell therapy.

In other embodiments, an additional agent is administered to subjects atthe same time as immune therapy as prophylaxis. In one embodiment theadditional agent comprises apoptotic cells, an apoptotic supernatant, aCTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereof oranalogue thereof, of a tellurium-based compound, or an immune-modulatingcompounds, or any combination thereof. In another embodiment, theadditional agent is administered to subjects 2-3 days afteradministration of immune therapy. In another embodiment, the additionalagent is administered to subjects 7 days after administration of immunetherapy. In another embodiment, the additional agent is administered tosubjects 10 days after administration of immune therapy. In anotherembodiment, the additional agent is administered to subjects 14 daysafter administration of immune therapy. In another embodiment, theadditional agent is administered to subjects 2-14 days afteradministration of immune therapy.

In another embodiment, the additional agent is administered to subjects2-3 hours after administration of immune therapy. In another embodiment,the additional agent is administered to subjects 7 hours afteradministration of immune therapy. In another embodiment the additionalagent is administered to subjects 10 hours after administration ofimmune therapy. In another embodiment, the additional agent isadministered to subjects 14 hours after administration of immunetherapy. In another embodiment, the additional agent is administered tosubjects 2-14 hours after administration of immune therapy.

In an alternative embodiment, the additional agent is administered tosubjects prior to immune therapy as prophylaxis. In another embodiment,the additional agent is administered to subjects 1 day beforeadministration of immune therapy. In another embodiment, the additionalagent is administered to subjects 2-3 days before administration ofimmune therapy. In another embodiment, the additional agent isadministered to subjects 7 days before administration of immune therapy.In another embodiment, the additional agent is administered to subjects10 days before administration of immune therapy. In another embodiment,the additional agent is administered to subjects 14 days beforeadministration of immune therapy. In another embodiment, the additionalagent is administered to subjects 2-14 days before administration ofimmune therapy.

In another embodiment, the additional agent is administered to subjects2-3 hours before administration of immune therapy. In anotherembodiment, the additional agent is administered to subjects 7 hoursbefore administration of immune therapy. In another embodiment, theadditional agent is administered to subjects 10 hours beforeadministration of immune therapy. In another embodiment, the additionalagent is administered to subjects 14 hours before administration ofimmune therapy. In another embodiment, the additional agent isadministered to subjects 2-14 hours before administration of immunetherapy.

In another embodiment, the additional agent is administeredtherapeutically, once cytokine release syndrome has occurred. In oneembodiment, the additional agent is administered once cytokine releaseleading up to or attesting to the beginning of cytokine release syndromeis detected. In one embodiment, the additional agent can terminate theincreased cytokine levels, or the cytokine release syndrome, and avoidits sequelae.

In another embodiment, the additional agent is administeredtherapeutically, at multiple time points. In another embodiment,administration of the additional agent is at least at two time pointsdescribed herein. In another embodiment, administration of theadditional agent is at least at three time points described herein. Inanother embodiment, administration of the additional agent is prior toCRS or a cytokine storm, and once cytokine release syndrome hasoccurred, and any combination thereof.

In one embodiment, the chimeric antigen receptor-expressing T-cell (CART-cell) therapy and the additional agent is administered together. Inanother embodiment, the CAR T-cell therapy is administered theadditional agent. In another embodiment, the CAR T-cell therapy isadministered prior to the additional agent. According to this aspect andin one embodiment, the additional agent is administered approximately2-3 weeks after the CAR T-cell therapy. In another embodiment, theadditional agent is administered approximately 6-7 weeks after the CART-cell therapy. In another embodiment, the additional agent isadministered approximately 9 weeks after the CAR T-cell therapy. Inanother embodiment, the additional agent is administered up to severalmonths after CAR T-cell therapy.

In one embodiment, CAR T-cells are heterologous to the subject. In oneembodiment, CAR T-cells are derived from one or more donors. In oneembodiment, CAR T-cells are derived from one or more bone marrow donors.In another embodiment, CAR T-cells are derived from one or more bloodbank donations. In one embodiment, the donors are matched donors. In oneembodiment, CAR T-cells are universal allogeneic CAR T-cells. In anotherembodiment, CAR T-cells are syngeneic CAR T-cells. In anotherembodiment, CAR T-cells are from unmatched third party donors. Inanother embodiment, CAR T-cells are from pooled third party donorT-cells. In one embodiment, the donor is a bone marrow donor. In anotherembodiment, the donor is a blood bank donor. In one embodiment, CART-cells of the compositions and methods as disclosed herein comprise oneor more MHC unrestricted tumor-directed chimeric receptors. In oneembodiment, non-autologous T-cells may be engineered or administeredaccording to protocols known in the art to prevent or minimizeautoimmune reactions, such as described in U.S. Patent Application No.20130156794, which is incorporated herein by references in its entirety.

In another embodiment, CAR T-cells are autologous to the subject. In oneembodiment, the patient's own cells are used. In this embodiment, if thepatient's own cells are used, then the CAR T-cell therapy isadministered after the apoptotic cell therapy.

In one embodiment, apoptotic cells are heterologous to the subject. Inone embodiment, apoptotic cells are derived from one or more donors. Inone embodiment, apoptotic cells are derived from one or more bone marrowdonors. In another embodiment, apoptotic cells are derived from one ormore blood bank donations. In one embodiment, the donors are matcheddonors. In another embodiment, apoptotic cells are from unmatched thirdparty donors. In one embodiment, apoptotic cells are universalallogeneic apoptotic cells. In another embodiment, apoptotic cells arefrom a syngeneic donor. In another embodiment, apoptotic cells are frompooled third party donor cells. In one embodiment, the donor is a bonemarrow donor. In another embodiment, the donor is a blood bank donor. Inanother embodiment, apoptotic cells are autologous to the subject. Inthis embodiment, the patient's own cells are used.

According to some embodiments, the therapeutic mononuclear-enriched cellpreparation disclosed herein or the apoptotic cell supernatant isadministered to the subject systemically. In another embodiment,administration is via the intravenous route. Alternately, thetherapeutic mononuclear enriched cell or supernatant may be administeredto the subject according to various other routes, including, but notlimited to, the parenteral, intraperitoneal, intra-articular,intramuscular and subcutaneous routes.

According to some embodiments, the therapeutic mononuclear-enriched cellpreparation disclosed herein or the additional agent is administered tothe subject systemically. In another embodiment, administration is viathe intravenous route. Alternately, the therapeutic mononuclear enrichedcell or the additional agent may be administered to the subjectaccording to various other routes, including, but not limited to, theparenteral, intraperitoneal, intra-articular, intramuscular andsubcutaneous routes.

In one embodiment, the preparation is administered in a local ratherthan systemic manner, for example, via injection of the preparationdirectly into a specific region of a patient's body. In anotherembodiment, a specific region comprises a tumor or cancer.

In another embodiment, the therapeutic mononuclear enriched cells orsupernatant are administered to the subject suspended in a suitablephysiological buffer, such as, but not limited to, saline solution, PBS,HBSS, and the like. In addition the suspension medium may furthercomprise supplements conducive to maintaining the viability of thecells. In another embodiment, the additional agent is administered tothe subject suspended in a suitable physiological buffer, such as, butnot limited to, saline solution, PBS, HBSS, and the like.

According to some embodiments the pharmaceutical composition isadministered intravenously. According to another embodiment, thepharmaceutical composition is administered in a single dose. Accordingto alternative embodiments the pharmaceutical composition isadministered in multiple doses. According to another embodiment, thepharmaceutical composition is administered in two doses. According toanother embodiment, the pharmaceutical composition is administered inthree doses. According to another embodiment, the pharmaceuticalcomposition is administered in four doses. According to anotherembodiment, the pharmaceutical composition is administered in five ormore doses. According to some embodiments, the pharmaceuticalcomposition is formulated for intravenous injection.

In one embodiment, any appropriate method of providing modifiedCAR-expressing immune cells to a subject can be used for methodsdescribed herein. In one embodiment, methods for providing cells to asubject comprise hematopoietic cell transplantation (HCT), infusion ofdonor-derived NK cells into cancer patients or a combination thereof.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering a compositioncomprising apoptotic cells to said subject.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering an apoptotic cellsupernatant, such as an apoptotic cell-phagocyte supernatant, to saidsubject.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering an at least oneadditional agent to said subject.

In certain embodiments, a CAR T-cell therapy comprises administering acomposition disclosed herein comprising CAR T-cells and either apoptoticcells or an apoptotic cell supernatant, or another or combination ofadditional agents as disclosed herein. In alternative embodiments, a CART-cell therapy comprises administering a composition disclosed hereincomprising CAR T-cells and a composition comprising either apoptoticcells or an apoptotic cell supernatant, or an additional agent orcombination thereof as disclosed herein.

Cytokine Release Associated with Non CAR T-Cell Applications

In one embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm, comprising the step of administering acomposition comprising apoptotic cells or an apoptotic supernatant tosaid subject, wherein said administering decreases or inhibits cytokineproduction in said subject. In another embodiment, decrease orinhibition of cytokine production is compared with a subjectexperiencing cytokine release syndrome or cytokine storm or vulnerableto cytokine release syndrome or cytokine storm and not administeredapoptotic cells or an apoptotic supernatant. In another embodiment,methods for decreasing or inhibiting cytokine production decrease orinhibit pro-inflammatory cytokine production. In another embodiment,methods for decreasing or inhibiting cytokine production decrease orinhibit production of at least one pro-inflammatory cytokine. In anotherembodiment, methods for decreasing or inhibiting cytokine productiondecrease or inhibit production of at least cytokine IL-6. In anotherembodiment, methods for decreasing or inhibiting cytokine productiondecrease or inhibit production of at least cytokine IL-1beta. In anotherembodiment, methods for decreasing or inhibiting cytokine productiondecrease or inhibit production of at least cytokine TNF-alpha. Inanother embodiment, methods disclosed herein for decreasing orinhibiting cytokine production, result in reduction or inhibition ofproduction of cytokines IL-6, IL-1β, or TNF-α, or any combination insaid subject compared with a subject experiencing cytokine releasesyndrome or cytokine storm or vulnerable to cytokine release syndrome orcytokine storm and not administered apoptotic cells or an apoptoticsupernatant.

Cancers or tumors may also affect the absolute level of cytokinesincluding pro-inflammatory cytokines. The level of tumor burden in asubject may affect cytokine levels, particularly proinflammatorycytokines. A skilled artisan would appreciate that the phrase “decreaseor inhibit” or grammatical variants thereof may encompass fold decreaseor inhibition of cytokine production, or a net decrease or inhibition ofcytokine production, or percent (%) decrease or inhibition, or mayencompass a rate of change of decrease or inhibition of a cytokineproduction.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm comprising the step of administeringapoptotic cells or a composition comprising apoptotic cells to saidsubject.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm comprising the step of administering anapoptotic cell supernatant, such as an apoptotic cell-phagocytesupernatant, or a composition comprising said supernatant to saidsubject.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm comprising the step of administering anapoptotic cell supernatant, such as an additional agent selected fromthe group comprising apoptotic cells, an apoptotic supernatant, a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof, or a composition comprising said supernatant tosaid subject.

In one embodiment, an infection causes the cytokine release syndrome orcytokine storm in the subject. In one embodiment, the infection is aninfluenza infection. In one embodiment, the influenza infection is H1N1.In another embodiment, the influenza infection is an H5N1 bird flu. Inanother embodiment, the infection is severe acute respiratory syndrome(SARS). In another embodiment, the subject has Epstein-Barrvirus-associated hemophagocytic lymphohistiocytosis (HLH). In anotherembodiment, the infection is sepsis. In one embodiment, the sepsis isgram-negative. In another embodiment, the infection is malaria. Inanother embodiment, the infection is an Ebola virus infection. Inanother embodiment, the infection is variola virus. In anotherembodiment, the infection is a systemic Gram-negative bacterialinfection. In another embodiment, the infection is Jarisch-Herxheimersyndrome.

In one embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is hemophagocytic lymphohistiocytosis (HLH).In another embodiment, HLH is sporadic HLH. In another embodiment, HLHis macrophage activation syndrome (MAS). In another embodiment, thecause of the cytokine release syndrome or cytokine storm in a subject isMAS.

In one embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is chronic arthritis. In another embodiment,the cause of the cytokine release syndrome or cytokine storm in asubject is systemic Juvenile Idiopathic Arthritis (sJIA), also known asStill's Disease.

In one embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is Cryopyrin-associated Periodic Syndrome(CAPS). In another embodiment, CAPS comprises Familial ColdAuto-inflammatory Syndrome (FCAS), also known as Familial Cold Urticaria(FCU). In another embodiment, CAPS comprises Muckle-Well Syndrome (MWS).In another embodiment, CAPS comprises Chronic Infantile NeurologicalCutaneous and Articular (CINCA) Syndrome. In yet another embodiment,CAPS comprises FCAS, FCU, MWS, or CINCA Syndrome, or any combinationthereof. In another embodiment, the cause of the cytokine releasesyndrome or cytokine storm in a subject is FCAS. In another embodiment,the cause of the cytokine release syndrome or cytokine storm in asubject is FCU. In another embodiment, the cause of the cytokine releasesyndrome or cytokine storm in a subject is MWS. In another embodiment,the cause of the cytokine release syndrome or cytokine storm in asubject is CINCA Syndrome. In still another embodiment, the cause of thecytokine release syndrome or cytokine storm in a subject is FCAS, FCU,MWS, or CINCA Syndrome, or any combination thereof.

In another embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is a cryopyrinopathy comprising inherited orde novo gain of function mutations in the NLRP3 gene, also known as theCIASI gene.

In one embodiment, the cause of the cytokine release syndrome orcytokine storm in a subject is a hereditary auto-inflammatory disorder.

In one embodiment, the trigger for the release of inflammatory cytokinesis a lipopolysaccharide (LPS), Gram-positive toxins, fungal toxins,glycosylphosphatidylinositol (GPI) or modulation of RIG-1 geneexpression.

In another embodiment, the subject experiencing cytokine releasesyndrome or cytokine storm does not have an infectious disease. In oneembodiment, the subject has acute pancreatitis. In another embodiment,the subject has tissue injury, which in on embodiment, is severe burnsor trauma. In another embodiment, the subject has acute respiratorydistress syndrome. In another embodiment, the subject has cytokinerelease syndrome or cytokine storm secondary to drug use. In anotherembodiment, the subject has cytokine release syndrome or cytokine stormsecondary to toxin inhalation.

In another embodiment, the subject has cytokine release syndrome orcytokine storm secondary to receipt of immunotherapy, which in oneembodiment is immunotherapy with superagonistic CD28-specific monoclonalantibodies (CD28SA). In one embodiment, the CD28SA is TGN1412. Inanother embodiment, the immunotherapy is CAR T-cell therapy.

In another embodiment, apoptotic cells or supernatant or a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof, may be used to control cytokine releasesyndrome or cytokine storm that results from administration of apharmaceutical composition. In one embodiment, the pharmaceuticalcomposition is oxaliplatin, cytarabine, lenalidomide, or a combinationthereof.

In another embodiment, apoptotic cells or the supernatant or a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof, may be used to control cytokine releasesyndrome or cytokine storm that results from administration of anantibody. In one embodiment, the antibody is monoclonal. In anotherembodiment, the antibody is polyclonal. In one embodiment, the antibodyis rituximab. In another embodiment, the antibody is Orthoclone OKT3(muromonab-CD3). In another embodiment, the antibody is alemtuzumab,tosituzumab, CP-870,893, LO-CD2a/BTI-322 or TGN1412.

In another embodiment, examples of diseases for which control ofinflammatory cytokine production can be beneficial include cancers,allergies, any type of infection, toxic shock syndrome, sepsis, any typeof autoimmune disease, arthritis, Crohn's disease, lupus, psoriasis, orany other disease for which the hallmark feature is toxic cytokinerelease that causes deleterious effects in a subject.

Sepsis

In some embodiments, disclosed herein is a method of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating sepsis in a subject in need, comprising the step ofadministering a composition comprising an early apoptotic cellpopulation to said subject, wherein said administering treats, prevents,inhibits, reduces the incidence of, ameliorates, or alleviates sepsis insaid subject. In some embodiments, disclosed herein is a method oftreating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need, comprising thestep of administering a composition comprising an apoptotic supernatantto said subject, wherein said administering treats, prevents, inhibits,reduces the incidence of, ameliorates, or alleviates sepsis in saidsubject.

In some embodiments, disclosed herein is a method of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating sepsis in a subject in need, comprising the step ofadministering a composition comprising an early apoptotic cellpopulation to said subject in combination with an antibiotic, whereinsaid administering treats, prevents, inhibits, reduces the incidence of,ameliorates, or alleviates sepsis in said subject. In some embodiments,disclosed herein is a method of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need, comprising the step of administering a compositioncomprising an apoptotic supernatant to said subject in combination withan antibiotic, wherein said administering treats, prevents, inhibits,reduces the incidence of, ameliorates, or alleviates sepsis in saidsubject.

In some embodiments, use of early apoptotic cells in the treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating of sepsis in a subject in need, is part of a combinationtherapy, for example but not limited to also administering to saidsubject an antibiotic.

In some embodiments, sepsis comprises severe sepsis. In someembodiments, sepsis comprises mild sepsis. In some embodiments, sepsiscomprises acute sepsis. In some embodiments, sepsis comprises highlyaggressive sepsis.

In some embodiments, the source of sepsis comprises pneumonia. In someembodiments, the source of sepsis comprises endovascularMethicillin-resistant Staphylococcus aureus (MRSA). In some embodiments,the source of sepsis comprises a urinary tract infection (UTI). In someembodiments, the source of sepsis comprises a biliary tract infection.

In some embodiments, treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needcomprises prevention, inhibiting, reducing the incidence of organfailure. In some embodiments, treating, preventing, inhibiting, reducingthe incidence of, ameliorating, or alleviating sepsis in a subject inneed comprises prevention, inhibiting, reducing the incidence of organdysfunction. In some embodiments, treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need comprises prevention, inhibiting, reducing the incidenceof organ failure. In some embodiments, treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need comprises prevention, inhibiting, reducing the incidenceof organ damage. In some embodiments, treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need comprises prevention, inhibiting, reducing the incidenceof acute multiple organ failure.

In some embodiments, administering early apoptotic cells to a subjectsuffering from sepsis results in preventing, inhibiting, reducing theincidence of organ failure. In some embodiments, administering earlyapoptotic cells to a subject suffering from sepsis results inpreventing, inhibiting, reducing the incidence of organ dysfunction. Insome embodiments, administering early apoptotic cells to a subjectsuffering from sepsis results in preventing inhibiting, reducing theincidence of organ failure. In some embodiments, administering earlyapoptotic cells to a subject suffering from sepsis results inpreventing, inhibiting, reducing the incidence of organ damage. In someembodiments, administering early apoptotic cells to a subject sufferingfrom sepsis results in preventing inhibiting, reducing the incidence ofacute multiple organ failure.

In some embodiments, organ failure during sepsis comprises failure of avital organ, for example but not limited to lung, heart, kidney, liver,and blood organs. In some embodiments, multiple organ failure as acomponent of sepsis comprises failure of a combination of lung, theheart, a kidney, liver, and blood. In some embodiments, hematologicalaberrations during sepsis comprise thrombocytopenia, lymphopenia,neutropenia, or neutrophilia, or any combination thereof. In someembodiments, organ failure may be measured using standards known in theart including but not limited to the Sequential Organ Failure Assessment(SOFA) scores. In some embodiments, measurements of sepsis use standardsknown in the art including but not limited to the Glasgow coma scale(GCS).

In some embodiments, treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needcomprises prevention, inhibiting, reducing the incidence of organdysfunction. In some embodiments, treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need comprises prevention, inhibiting, reducing the incidenceof multiple organ dysfunction. In some embodiments, treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating sepsis in a subject in need comprises prevention,inhibiting, reducing the incidence of vital organ dysfunction.

In some embodiments, administering early apoptotic cells to a subjectsuffering from sepsis results in preventing, inhibiting, reducing theincidence of organ dysfunction. In some embodiments, administering earlyapoptotic cells to a subject suffering from sepsis results inpreventing, inhibiting, reducing the incidence of multiple organdysfunction. In some embodiments, administering early apoptotic cells toa subject suffering from sepsis results in preventing, inhibiting,reducing the incidence of vital organ dysfunction. In some embodiments,administering early apoptotic cells to a subject suffering from sepsisresults in preventing an increase in vital organ dysfunction, comparedwith subjects not administered early apoptotic cells.

In some embodiments, administering early apoptotic cells to a subjectsuffering from sepsis is highly effective in the treatment of sepsis. Insome embodiments, measure of an effective treatment of sepsis includesthe percent of patients that recover from sepsis within a giventimeframe. In some embodiments, measure of an effective treatment ofsepsis includes the percent of patients that are released from intensivecare compared with the percent of patients not administered earlyapoptotic cells. In some embodiments, a subject suffering from sepsisadministered early apoptotic cells recovers more quickly than a subjectsuffering from sepsis and not administered early apoptotic cells. Insome embodiments, a subject suffering from sepsis administered earlyapoptotic cells recovers more completely than a subject suffering fromsepsis and not administered early apoptotic cells. In some embodiments,the mortality rate of patients suffering from sepsis and treated withearly apoptotic cells is decreased, compared with patients notadministered early apoptotic cells.

In some embodiments, treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needcomprises prevention, inhibiting, reducing the incidence ofcardiovascular dysfunction. In some embodiments, treating, preventing,inhibiting, reducing the incidence of, ameliorating, or alleviatingsepsis in a subject in need comprises prevention, inhibiting, reducingthe incidence of acute kidney injury. In some embodiments, treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating sepsis in a subject in need comprises prevention,inhibiting, reducing the incidence of lung dysfunction. In someembodiments, treating, preventing, inhibiting, reducing the incidenceof, ameliorating, or alleviating sepsis in a subject in need comprisesprevention, inhibiting, reducing the incidence of liver dysfunction. Insome embodiments, treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needcomprises prevention, inhibiting, reducing the incidence ofhematological aberrations. In some embodiments, treating, preventing,inhibiting, reducing the incidence of, ameliorating, or alleviatingsepsis in a subject in need comprises prevention, inhibiting, reducingthe incidence of a combination of any of cardiovascular dysfunction,acute kidney injury, lung dysfunction, and hematological aberrations.

In some embodiments, administering early apoptotic cells to a subjectsuffering from sepsis results in preventing, inhibiting, reducing theincidence of cardiovascular dysfunction. In some embodiments,administering early apoptotic cells to a subject suffering from sepsisresults in preventing, inhibiting, reducing the incidence of acutekidney injury. In some embodiments, administering early apoptotic cellsto a subject suffering from sepsis results in preventing, inhibiting,reducing the incidence of lung dysfunction. In some embodiments,administering early apoptotic cells to a subject suffering from sepsisresults in preventing, inhibiting, reducing the incidence of liverdysfunction. In some embodiments, administering early apoptotic cells toa subject suffering from sepsis results in preventing, inhibiting,reducing the incidence of hematological aberrations. In someembodiments, administering early apoptotic cells to a subject sufferingfrom sepsis results in preventing, inhibiting, reducing the incidence ofa combination of any of cardiovascular dysfunction, acute kidney injury,lung dysfunction, and hematological aberrations.

In some embodiments, treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needcomprises prevention, inhibiting, reducing the incidence of a cytokinestorm. In some embodiments, treating, preventing, inhibiting, reducingthe incidence of, ameliorating, or alleviating sepsis in a subject inneed comprises prevention, inhibiting, reducing the incidence of achemokine storm. In some embodiments, treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need comprises prevention, inhibiting, reducing the incidenceof a cytokine and chemokine storm.

In some embodiments, administering early apoptotic cells to a subjectsuffering from sepsis results in preventing, inhibiting, reducing theincidence of a cytokine storm. In some embodiments, administering earlyapoptotic cells to a subject suffering from sepsis results inpreventing, inhibiting, reducing the incidence of a chemokine storm. Insome embodiments, administering early apoptotic cells to a subjectsuffering from sepsis results in preventing, inhibiting, reducing theincidence of a cytokine and chemokine storm.

In some embodiments, treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needcomprises rebalancing the immune response in a subject. In someembodiments, treating, preventing, inhibiting, reducing the incidenceof, ameliorating, or alleviating sepsis in a subject in need comprisesreducing secretion of pro-inflammatory cytokines. In some embodiments,treating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need comprisesreducing secretion of pro-inflammatory cytokines/chemokines andanti-inflammatory cytokines/chemokines.

In some embodiments, administering early apoptotic cells to a subjectsuffering from sepsis results in rebalancing the immune response in asubject. In some embodiments, administering early apoptotic cells to asubject suffering from sepsis results in reducing secretion ofpro-inflammatory cytokines. In some embodiments, administering earlyapoptotic cells to a subject suffering from sepsis results in reducingsecretion of pro-inflammatory cytokines/chemokines and anti-inflammatorycytokines/chemokines.

In some embodiments, treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needcomprises a reduction in mortality of a subject suffering from sepsis.In some embodiments, treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needcomprises improving the survival time in the subject in need.

In some embodiments, method of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need increase the survival time in said subject by greaterthan 60% compared with a subject not administered apoptotic cells. Insome embodiments, method of treating, preventing, inhibiting, reducingthe incidence of, ameliorating, or alleviating sepsis in a subject inneed increase the survival time in said subject by greater than 70%compared with a subject not administered apoptotic cells. In someembodiments, method of treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needincrease the survival time in said subject by greater than 80% comparedwith a subject not administered apoptotic cells. In some embodiments,method of treating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need increase thesurvival time in said subject by greater than 90% compared with asubject not administered apoptotic cells. In some embodiments, method oftreating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need increase thesurvival time in said subject by greater than 100% compared with asubject not administered apoptotic cells.

In some embodiments, method of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need increase the survival time in said subject by about50%-100%, compared with a subject not administered apoptotic cells. Insome embodiments, method of treating, preventing, inhibiting, reducingthe incidence of, ameliorating, or alleviating sepsis in a subject inneed increase the survival time in said subject by about 80%-100%,compared with a subject not administered apoptotic cells. In someembodiments, method of treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needincrease the survival time in said subject by about 80%, 90%, or 100%compared with a subject not administered apoptotic cells.

In some embodiments, method of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need increase the survival time in said subject by about100%-2000%, compared with a subject not administered apoptotic cells. Insome embodiments, method of treating, preventing, inhibiting, reducingthe incidence of, ameliorating, or alleviating sepsis in a subject inneed increase the survival time in said subject by about 200%-300%,compared with a subject not administered apoptotic cells. In someembodiments, method of treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needincrease the survival time in said subject by greater than 100% comparedwith a subject not administered apoptotic cells. In some embodiments,method of treating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need increase thesurvival time in said subject by greater than 200% compared with asubject not administered apoptotic cells. In some embodiments, method oftreating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need increase thesurvival time in said subject by greater than 300% compared with asubject not administered apoptotic cells. In some embodiments, method oftreating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need increase thesurvival time in said subject by greater than 400% compared with asubject not administered apoptotic cells. In some embodiments, method oftreating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need increase thesurvival time in said subject by greater than 500%, 600%, 700%, 800%,900%, or 1000% compared with a subject not administered apoptotic cells.

In some embodiments, method of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need increase the survival time in said subject by about 100%compared with a subject not administered apoptotic cells. In someembodiments, method of treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needincrease the survival time in said subject by about 200%, 300%, 400%,500%, 600%, 700%, 800%, 900%, or 1000%, compared with a subject notadministered apoptotic cells.

In some embodiments, method of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need increase the survival time in said subject by about100%-1000%, compared with a subject not administered apoptotic cells. Insome embodiments, method of treating, preventing, inhibiting, reducingthe incidence of, ameliorating, or alleviating sepsis in a subject inneed increase the survival time in said subject by about 100%-500%,compared with a subject not administered apoptotic cells. In someembodiments, method of treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needincrease the survival time in said subject by about 500%-1000%, comparedwith a subject not administered apoptotic cells. In some embodiments,method of treating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need increase thesurvival time in said subject by about 70%-80%, compared with a subjectnot administered apoptotic cells. In some embodiments, method oftreating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating sepsis in a subject in need increase thesurvival time in said subject by about 50% compared with a subject notadministered apoptotic cells. In some embodiments, method of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating sepsis in a subject in need increase the survival time insaid subject by about 60% compared with a subject not administeredapoptotic cells. In some embodiments, method of treating, preventing,inhibiting, reducing the incidence of, ameliorating, or alleviatingsepsis in a subject in need increase the survival time in said subjectby about 70% compared with a subject not administered apoptotic cells.In some embodiments, method of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating sepsis in asubject in need increase the survival time in said subject by about 80%compared with a subject not administered apoptotic cells.

In another embodiment, treating, preventing, inhibiting, reducing theincidence of, ameliorating, or alleviating sepsis in a subject in needis compared with a subject experiencing sepsis and not administeredapoptotic cells. In another embodiment, treating, preventing,inhibiting, reducing the incidence of, ameliorating, or alleviatingsepsis in a subject in need is compared with a subject experiencingsepsis and not administered an apoptotic supernatant.

In some embodiments, administration of apoptotic cells to a subjectexperiencing sepsis comprises intravenous administration. In someembodiments, administration of apoptotic cells to a subject experiencingsepsis comprising intravenous administration following an initialstandard of care treatment with antibiotics, fluids, and vasopressors.

In some embodiments, administration of apoptotic cells to a subjectexperiencing sepsis comprises administration between 12-24 hours postdiagnosis of sepsis. In some embodiments, administration of apoptoticcells to a subject experiencing sepsis comprises administration between12-36 hours post diagnosis of sepsis. In some embodiments,administration of apoptotic cells to a subject experiencing sepsiscomprises administration between 24-36 hours post diagnosis of sepsis.In some embodiments, administration of apoptotic cells to a subjectexperiencing sepsis comprises administration between 12-18 hours postdiagnosis of sepsis. In some embodiments, administration of apoptoticcells to a subject experiencing sepsis comprises administration between18-24 hours post diagnosis of sepsis. In some embodiments,administration of apoptotic cells to a subject experiencing sepsiscomprises administration between 18-30 hours post diagnosis of sepsis.In some embodiments, administration of apoptotic cells to a subjectexperiencing sepsis comprises administration between 24-30 hours postdiagnosis of sepsis. In some embodiments, administration of apoptoticcells to a subject experiencing sepsis comprises administration between24-36 hours post diagnosis of sepsis.

In some embodiments, administration of apoptotic cells to a subjectexperiencing sepsis comprises administration about 12 hours postdiagnosis of sepsis. In some embodiments, administration of apoptoticcells to a subject experiencing sepsis comprises administration about13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, or hours post diagnosis of sepsis. In someembodiments, administration of apoptotic cells to a subject experiencingsepsis comprises administration within 24 hours±6 hours post diagnosisof sepsis.

In some embodiments, the response of a subject suffering sepsis andadministered a composition comprising apoptotic cells comprises a doseresponse. In some embodiments, the response of a subject sufferingsepsis and administered a composition comprising an apoptotic cellsupernatant comprises a dose response.

Alpha-1-Antitrypsin (AAT)

Alpha-1-antitrypsin (AAT) is a circulating 52-kDa glycoprotein that isproduced mainly by the liver. AAT is primarily known as a serineprotease inhibitor and is encoded by the gene SERPINA1. AAT inhibitsneutrophil elastase, and inherited deficiency in circulating AAT resultsin lung-tissue deterioration and liver disease. Serum AAT concentrationsin healthy individuals increase twofold during inflammation.

There is a negative association between AAT levels and the severity ofseveral inflammatory diseases. For example, reduced levels or activityof AAT have been described in patients with HIV infection, diabetesmellitus, hepatitis C infection-induced chronic liver disease, andseveral types of vasculitis.

Increasing evidence demonstrates that human serum derivedalpha-1-anti-trypsin (AAT) reduces production of pro-inflammatorycytokines, induces anti-inflammatory cytokines, and interferes withmaturation of dendritic cells.

Indeed, the addition of AAT to human peripheral blood mononuclear cells(PBMC) inhibits LPS induced release of TNF-α and IL-1β but increasesIL-1 receptor antagonist (IL-1Ra) and IL-10 production.

AAT reduces in vitro IL-1β-mediated pancreatic islet toxicity, and AATmonotherapy prolongs islet allograft survival, promotes antigen-specificimmune tolerance in mice, and delays the development of diabetes innon-obese diabetic (NOD) mice. AAT was shown to inhibit LPS-inducedacute lung injury in experimental models. Recently, AAT was shown toreduce the size of infarct and the severity of heart failure in a mousemodel of acute myocardial ischemia-reperfusion injury.

Monotherapy with clinical-grade human AAT (hAAT) reduced circulatingpro-inflammatory cytokines, diminished Graft vs Host Disease (GvHD)severity, and prolonged animal survival after experimental allogeneicbone marrow transfer (Tawara et al., Proc Natl Acad Sci USA. 2012 Jan.10; 109(2):564-9), incorporated herein by reference. AAT treatmentreduced the expansion of alloreactive T effector cells but enhanced therecovery of T regulatory T-cells, (Tregs) thus altering the ratio ofdonor T effector to T regulatory cells in favor of reducing thepathological process. In vitro, AAT suppressed LPS-induced in vitrosecretion of proinflammatory cytokines such as TNF-α and IL-1β, enhancedthe production of the anti-inflammatory cytokine IL-10, and impairedNF-κB translocation in the host dendritic cells. Marcondes, Blood. 2014(Oct. 30; 124(18):2881-91) incorporated herein by reference show thattreatment with AAT not only ameliorated GvHD but also preserved andperhaps even enhanced the graft vs leukemia (GVL) effect.

In one embodiment, disclosed herein are compositions comprising chimericantigen receptor-expressing T-cells (CAR T-cells) andAlpha-1-antitrypsin (AAT). In another embodiment, CAR T-cells andAlpha-1-antitrypsin (AAT) are in separate compositions. In anotherembodiment, AAT comprises a full length AAT or a functional fragmentthereof. In another embodiment, AA comprises an analogue of a fulllength AAT or a functional fragment thereof. In another embodiment, acomposition comprising AAT further comprises apoptotic cells or anapoptotic cell supernatant.

In another embodiment, disclosed herein is a method of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor in a subject comprising the step ofadministering chimeric antigen receptor-expressing T-cells (CAR T-cells)and a composition comprising Alpha-1-antitrypsin (AAT) to said subject.In another embodiment, the method further comprises apoptotic cells oran apoptotic cell supernatant.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering a compositioncomprising Alpha-1-antitrypsin (AAT) to said subject. In anotherembodiment, a method of treating cytokine release syndrome or a cytokinestorm in a subject undergoing chimeric antigen receptor-expressingT-cell (CAR T-cell) therapy, comprises the step of administering acomposition comprising Alpha-1-antitrypsin (AAT) to said subject. Inanother embodiment, a method of preventing cytokine release syndrome ora cytokine storm in a subject undergoing chimeric antigenreceptor-expressing T-cell (CAR T-cell) therapy, comprises the step ofadministering a composition comprising Alpha-1-antitrypsin (AAT) to saidsubject. In another embodiment, a method of ameliorating cytokinerelease syndrome or a cytokine storm in a subject undergoing chimericantigen receptor-expressing T-cell (CAR T-cell) therapy, comprises thestep of administering a composition comprising Alpha-1-antitrypsin (AAT)to said subject. In another embodiment, a method of alleviating cytokinerelease syndrome or a cytokine storm in a subject undergoing chimericantigen receptor-expressing T-cell (CAR T-cell) therapy, comprises thestep of administering a composition comprising Alpha-1-antitrypsin (AAT)to said subject.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm, comprising the step of administering acomposition comprising Alpha-1-antitrypsin (AAT) to said subject.

In one embodiment, AAT is administered alone to control cytokinerelease. In another embodiment, both AAT and apoptotic cells or acomposition thereof, or apoptotic cell supernatants or a compositionthereof, are administered to control cytokine release.

Immuno-Modulatory Agents

A skilled artisan would appreciate that immune-modulating agents mayencompass extracellular mediators, receptors, mediators of intracellularsignaling pathways, regulators of translation and transcription, as wellas immune cells. In one embodiment, an additional agent disclosed hereinis an immune-modulatory agent known in the art. In another embodiment,use in the methods disclosed here of an immune-modulatory agent reducesthe level of at least one cytokine. In another embodiment, use in themethods disclosed here of an immune-modulatory agent reduces or inhibitsCRS or a cytokine storm. In some embodiments, use in the methodsdisclosed herein of an immune-modulatory agent is for treating,preventing, inhibiting the growth, delaying disease progression,reducing the tumor load, or reducing the incidence of a tumor or acancer, or any combination thereof. In some embodiments, use of animmune-modulatory agent is in combination with another compositiondisclosed herein, for example but not limited to a compositioncomprising early apoptotic cells or comprising CAR T-cells.

In one embodiment, an immune-modulatory agent comprises compounds thatblock, inhibit or reduce the release of cytokines or chemokines. Inanother embodiment, an immune-modulatory agent comprises compounds thatblock, inhibit or reduce the release of IL-21 or IL-23, or a combinationthereof. In another embodiment, an immune-modulatory agent comprises anantiretroviral drug in the chemokine receptor-5 (CCR5) receptorantagonist class, for example maraviroc. In another embodiment, animmune-modulatory agent comprises an anti-DNAM-1 antibody. In anotherembodiment, an immune-modulatory agent comprisesdamage/pathogen-associated molecules (DAMPs/PAMPs) selected from thegroup comprising heparin sulfate, ATP, and uric acid, or any combinationthereof. In another embodiment, an immune-modulatory agent comprises asialic acid binding Ig-like lectin (Siglecs). In another embodiment, animmune-modulatory agent comprises a cellular mediator of tolerance, forexample regulatory CD4⁺ CD25⁺ T cells (Tregs) or invariant naturalkiller T cells (iNK T-cells). In another embodiment, animmune-modulatory agent comprises dendritic cells. In anotherembodiment, an immune-modulatory agent comprises monocytes. In anotherembodiment, an immune-modulatory agent comprises macrophages. In anotherembodiment, an immune-modulatory agent comprises JAK2 or JAK3 inhibitorsselected from the group comprising ruxolitinib and tofacitinib. Inanother embodiment, an immune-modulatory agent comprises an inhibitor ofspleen tyrosine kinase (Syk), for example fostamatinib. In anotherembodiment, an immune-modulatory agent comprises histone deacetylaseinhibitor vorinostat acetylated STAT3. In another embodiment, animmune-modulatory agent comprises neddylation inhibitors, for exampleMLN4924. In another embodiment, an immune-modulatory agent comprises anmiR-142 antagonist. In another embodiment, an immune-modulatory agentcomprises a chemical analogue of cytidine, for example Azacitidine. Inanother embodiment, an immune-modulatory agent comprises an inhibitor ofhistone deacetylase, for example Vorinostat. In another embodiment, animmune-modulatory agent comprises an inhibitor of histone methylation.In another embodiment, an immune-modulatory agent comprises an antibody.In another embodiment, the antibody is rituximab (RtX)

Tellurium-Based Compounds

Tellurium is a trace element found in the human body. Various telluriumcompounds, have immune-modulating properties, and have been shown tohave beneficial effects in diverse preclinical and clinical studies. Aparticularly effective family of tellurium-containing compounds isdisclosed for example, in U.S. Pat. Nos. 4,752,614; 4,761,490; 4,764,461and 4,929,739. The immune-modulating properties of this family oftellurium-containing compounds is described, for example, in U.S. Pat.Nos. 4,962,207, 5,093,135, 5,102,908 and 5,213,899, which are allincorporated by reference as if fully set forth herein.

One promising compound is ammoniumtrichloro(dioxyethylene-O,O′)tellurate, which is also referred to hereinand in the art as AS101. AS101, as a representative example of thefamily of tellurium-containing compound discussed hereinabove, exhibitsantiviral (Nat. Immun. Cell Growth Regul. 7(3):163-8, 1988; AIDS Res HumRetroviruses. 8(5):613-23, 1992), and tumoricidal activity (Nature330(6144):173-6, 1987; J. Clin. Oncol. 13(9):2342-53, 1995; J. Immunol.161(7):3536-42, 1998). Further, AS101 is characterized by low toxicity.

In one embodiment, a composition comprising tellurium-containingimmune-modulator compounds may be used in methods disclosed herein,where the tellurium-based compound stimulates the innate and acquiredarm of the immune response. For example, it has been shown that AS101 isa potent activator of interferon (IFN) in mice (J. Natl. Cancer Inst.88(18):1276-84, 1996) and humans (Nat. Immun. Cell Growth Regul.9(3):182-90, 1990; Immunology 70(4):473-7, 1990; J. Natl. Cancer Inst.88(18):1276-84, 1996.)

In another embodiment, tellurium-based compounds induce the secretion ofa spectrum of cytokines, such as IL-1α, IL-6 and TNF-α.

In another embodiment, a tellurium-based compound comprises atellurium-based compound known in the art to have immune-modulatingproperties. In another embodiment, a tellurium-based compound comprisesammonium trichloro(dioxyethylene-O,O′)tellurate.

In one embodiment, a tellurium-based compound inhibits the secretion ofat least one cytokine. In another embodiment, a tellurium-based compoundreduces the secretion of at least one cytokine. In another embodiment, atellurium-based compound inhibits or reduces a cytokine release syndrome(CRS) of a cytokine storm.

In one embodiment, disclosed herein are compositions comprising chimericantigen receptor-expressing T-cells (CAR T-cells) and a tellurium-basedcompound. In another embodiment, CAR T-cells and Tellurium-basedcompound are in separate compositions. In another embodiment, AATcomprises a full length AAT or a functional fragment thereof. In anotherembodiment, AA comprises an analogue of a full length AAT or afunctional fragment thereof

In another embodiment, disclosed herein is a method of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor in a subject comprising the step ofadministering chimeric antigen receptor-expressing T-cells (CAR T-cells)and a composition comprising a Tellurium-based compound to said subject.

In another embodiment, disclosed herein is a method of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing chimeric antigen receptor-expressing T-cell (CART-cell) therapy, comprising the step of administering a compositioncomprising a Tellurium-based compound to said subject. In anotherembodiment, a method of treating cytokine release syndrome or a cytokinestorm in a subject undergoing chimeric antigen receptor-expressingT-cell (CAR T-cell) therapy, comprises the step of administering acomposition comprising a Tellurium-based compound to said subject. Inanother embodiment, a method of preventing cytokine release syndrome ora cytokine storm in a subject undergoing chimeric antigenreceptor-expressing T-cell (CAR T-cell) therapy, comprises the step ofadministering a composition comprising a Tellurium-based compound tosaid subject. In another embodiment, a method of ameliorating cytokinerelease syndrome or a cytokine storm in a subject undergoing chimericantigen receptor-expressing T-cell (CAR T-cell) therapy, comprises thestep of administering a composition comprising a Tellurium-basedcompound to said subject. In another embodiment, a method of alleviatingcytokine release syndrome or a cytokine storm in a subject undergoingchimeric antigen receptor-expressing T-cell (CAR T-cell) therapy,comprises the step of administering a composition comprising aTellurium-based compound to said subject.

In another embodiment, disclosed herein is a method of decreasing orinhibiting cytokine production in a subject experiencing cytokinerelease syndrome or cytokine storm or vulnerable to cytokine releasesyndrome or cytokine storm, comprising the step of administering acomposition comprising a Tellurium-based compound to said subject.

In one embodiment, a tellurium-based compound is administered alone tocontrol cytokine release. In another embodiment, both a tellurium-basedcompound and apoptotic cells or a composition thereof, or apoptotic cellsupernatants or a composition thereof, are administered to controlcytokine release.

Dendritic Cells

In one embodiment, dendritic cells (DCs) are antigen-producing andpresenting cells of the mammalian immune system that process antigenmaterial and present it on the cell surface to the T-cells of the immunesystem and are thereby capable of sensitizing T-cells to both new andrecall antigens. In another embodiment, DCs are the most potentantigen-producing cells, acting as messengers between the innate and theadaptive immune systems. DC cells may be used, in one embodiment, toprime specific antitumor immunity through the generation of effectorcells that attack and lyse tumors.

Dendritic cells are present in those tissues that are in contact withthe external environment, such as the skin (where there is a specializeddendritic cell type called the Langerhans cell) and the inner lining ofthe nose, lungs, stomach and intestines. They can also be found in animmature state in the blood. Once activated, they migrate to the lymphnodes where they interact with T-cells and B cells to initiate and shapethe adaptive immune response. At certain development stages, they growbranched projections, the dendrites that give the cell its name.Dendritic cells may be engineered to express particular tumor antigens.

The three signals that are required for T-cell activation are: (i)presentation of cognate antigen in self MHC molecules; (ii)costimulation by membrane-bound receptor-ligand pairs; and (iii) solublefactors to direct polarization of the ensuing immune response. Dendriticcells (DCs) are able to provide all of the three signals required forT-cell activation making them an excellent cancer vaccine platform.

Therefore, in one embodiment, disclosed herein are a compositioncomprising dendritic cells and an additional agent, wherein saidadditional agent comprises apoptotic cells, apoptotic supernatants, aCTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereof oranalogue thereof, a tellurium-based compound, or an immune modulatingagent, or any combination thereof.

In another embodiment, disclosed herein is a method of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor in a subject comprising the step ofadministering dendritic cells and a composition comprising an additionalagent, wherein said agent comprises apoptotic cells, apoptoticsupernatants, a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment thereof or analogue thereof, a tellurium-based compound, or animmune modulating agent, or any combination thereof, to said subject.

Genetic Modification

In some embodiments, genetic modification of T-cells, dendritic cells,and/or apoptotic cells may be accomplished using RNA, DNA, recombinantviruses, or a combination thereof. In some embodiments, vectors derivedfrom gamma retroviruses or lentiviruses are used in the compositions andmethods as disclosed herein. In another embodiment, these vectors canintegrate into the host genome, with potentially permanent expression ofthe transgene and have low intrinsic immunogenicity. In anotherembodiment, another vector that integrates into the host genome and/orhas low intrinsic immunogenicity may be used in the compositions andmethods as disclosed herein. In another embodiment, thenon-viral-vector-mediated sleeping beauty transposon system is used toinsert the CAR and other genes into the T-cell. In another embodiment,“suicide genes” are integrated into the T-cells, in which expression ofa pro-apoptotic gene is under the control of an inducible promoterresponsive to a systemically delivered drug.

In some embodiments, genetic modification may be transient. In anotherembodiment, genetic modification may utilize messenger RNA (mRNA). Inanother embodiment, large numbers of cells may be infused on multipleoccasions in transiently engineered T-cells, such as mRNA-transfectedT-cells. In another embodiment, RNA-based electroporation of lymphocytesusing in vitro-transcribed mRNA mediates transient expression ofproteins for approximately one week and obviates the risk of integratingviral vectors. In another embodiment, mRNA-transduced dendritic cells ormRNA-electroporated T and NK lymphocytes.

It has been demonstrated that genetically modified T-cells can persistafter adoptive transfer for more than a decade without adverse effects,indicating that genetically modifying human T-cells is fundamentallysafe.

In another embodiment, the genetic modification of the compositions andin the methods as disclosed herein may be any method that is known inthe art.

Apoptotic Cells

Production of apoptotic cells (“ApoCells”) for use in compositions andmethods as disclosed herein, has been described in WO 2014/087408, whichis incorporated by reference herein in its entirety, and is described inbrief in Example 1 below. In another embodiment, apoptotic cells for usein compositions and methods as disclosed herein are produced in any waythat is known in the art. In another embodiment, apoptotic cells for usein compositions and methods disclosed herein are autologous with asubject undergoing therapy. In another embodiment, apoptotic cells foruse in compositions and methods disclosed herein are allogeneic with asubject undergoing therapy. In another embodiment, a compositioncomprising apoptotic cells comprises apoptotic cells as disclosed hereinor as is known in art.

A skilled artisan would appreciate that the term “autologous” mayencompass a tissue, cell, nucleic acid molecule or polypeptide in whichthe donor and recipient is the same person.

A skilled artisan would appreciate that the term “allogeneic” mayencompass a tissue, cell, nucleic acid molecule or polypeptide that isderived from separate individuals of the same species. In someembodiments, allogeneic donor cells are genetically distinct from therecipient.

In some embodiments, obtaining a mononuclear-enriched cell compositionaccording to the production method disclosed herein is effected byleukapheresis. A skilled artisan would appreciate that the term“leukapheresis” may encompass an apheresis procedure in which leukocytesare separated from the blood of a donor. In some embodiments, the bloodof a donor undergoes leukapheresis and thus a mononuclear-enriched cellcomposition is obtained according to the production method disclosedherein. It is to be noted, that the use of at least one anticoagulantduring leukapheresis is required, as is known in the art, in order toprevent clotting of the collected cells.

In some embodiments, the leukapheresis procedure is configured to allowcollection of mononuclear-enriched cell composition according to theproduction method disclosed herein. In some embodiments, cellcollections obtained by leukapheresis comprise at least 65%. In otherembodiments, at least 70%, or at least 80% mononuclear cells. asdisclosed herein. In some embodiments, blood plasma from the cell-donoris collected in parallel to obtaining of the mononuclear-enriched cellcomposition In the production method disclosed herein. In someembodiments, about 300-600 ml of blood plasma from the cell-donor arecollected in parallel to obtaining the mononuclear-enriched cellcomposition according to the production method disclosed herein. In someembodiments, blood plasma collected in parallel to obtaining themononuclear-enriched cell composition according to the production methoddisclosed herein is used as part of the freezing and/or incubationmedium. Additional detailed methods of obtaining an enriched populationof apoptotic cells for use in the compositions and methods as disclosedherein may be found in WO 2014/087408, which is incorporated herein byreference in its entirety.

In some embodiments, the early apoptotic cells for use in the methodsdisclosed herein comprise at least 85% mononuclear cells. In furtherembodiments, the early apoptotic cells for use in the methods disclosedherein contains at least 85% mononuclear cells, 90% mononuclear cells oralternatively over 90% mononuclear cells. In some embodiments, the earlyapoptotic cells for use in the methods disclosed herein comprise atleast 90% mononuclear cells. In some embodiments, the early apoptoticcells for use in the methods disclosed herein comprise at least 95%mononuclear cells.

It is to be noted that, in some embodiments, while themononuclear-enriched cell preparation at cell collection comprises atleast 65%, preferably at least 70%, most preferably at least 80%mononuclear cells, the final pharmaceutical population, following theproduction method of the early apoptotic cells for use in the methodsdisclosed herein, comprises at least 85%, preferably at least 90%, mostpreferably at least 95% mononuclear cells.

In certain embodiments, the mononuclear-enriched cell preparation usedfor production of the composition of the early apoptotic cells for usein the methods disclosed herein comprises at least 50% mononuclear cellsat cell collection. In certain embodiments, disclosed herein is a methodfor producing the pharmaceutical population wherein the method comprisesobtaining a mononuclear-enriched cell preparation from the peripheralblood of a donor, the mononuclear-enriched cell preparation comprisingat least 50% mononuclear cells. In certain embodiments, disclosed hereinis a method for producing the pharmaceutical population wherein themethod comprises freezing a mononuclear-enriched cell preparationcomprising at least 50% mononuclear cells.

In some embodiments, the cell preparation comprises at least 85%mononuclear cells, wherein at least 40% of the cells in the preparationare in an early-apoptotic state, wherein at least 85% of the cells inthe preparation are viable cells. In some embodiments, the apoptoticcell preparation comprises no more than 15% CD15^(high) expressingcells.

A skilled artisan would appreciate that the term “early-apoptotic state”may encompass cells that show early signs of apoptosis without latesigns of apoptosis. Examples of early signs of apoptosis in cellsinclude exposure of phosphatidylserine (PS) and the loss ofmitochondrial membrane potential. Examples of late events includepropidium iodide (PI) admission into the cell and the final DNA cutting.In order to document that cells are in an “early apoptotic” state, insome embodiments, PS exposure detection by Annexin-V and PI staining areused, and cells that are stained with Annexin V but not with PI areconsidered to be “early apoptotic cells”. In another embodiment, cellsthat are stained by both Annexin-V FITC and PI are considered to be“late apoptotic cells”. In another embodiment, cells that do not stainfor either Annexin-V or PI are considered non-apoptotic viable cells.

A skilled artisan would appreciate that in some embodiments the terms“early apoptotic cells”, “apoptotic cell”, “Allocetra”, “ALC”, and“ApoCell”, and grammatical variants thereof, may be used interchangeablyhaving all the same qualities and meanings. The skilled artisan wouldappreciate that the compositions and methods described herein, in someembodiments comprise early apoptotic cells. In some embodiments, asdescribed herein, early apoptotic cells are HLA matched to a recipientof a composition comprising the early apoptotic cells (a subject inneed). In some embodiments, as described herein, early apoptotic cellsare not matched to a recipient of a composition comprising the earlyapoptotic cells (a subject in need). In some embodiments, the earlyapoptotic cells not matched to a recipient of a composition comprisingthe early apoptotic cells (a subject in need) are irradiated asdescribed herein in detail. In some embodiments, irradiated not matchedcells are termed “Allocetra-OTS” or “ALC-OTS”.

In some embodiments, apoptotic cells comprise cells in an earlyapoptotic state. In another embodiment, apoptotic cells comprise cellswherein at least 90% of said cells are in an early apoptotic state. Inanother embodiment, apoptotic cells comprise cells wherein at least 80%of said cells are in an early apoptotic state. In another embodiment,apoptotic cells comprise cells wherein at least 70% of said cells are inan early apoptotic state. In another embodiment, apoptotic cellscomprise cells wherein at least 60% of said cells are in an earlyapoptotic state. In another embodiment, apoptotic cells comprise cellswherein at least 50% of said cells are in an early apoptotic state.

In some embodiments, the composition comprising apoptotic cells furthercomprises an anti-coagulant.

In some embodiments, early apoptotic cells are stable. A skilled artisanwould appreciate that in some embodiments stability encompassesmaintaining early apoptotic cell characteristics over time, for example,maintaining early apoptotic cell characteristics upon storage at about2-8° C. In some embodiments, stability comprises maintaining earlyapoptotic cell characteristic upon storage at freezing temperatures, forexample temperatures at or below 0° C.

In some embodiments, the mononuclear-enriched cell population obtainedaccording to the production method of the early apoptotic cells for usein the methods disclosed herein undergoes freezing in a freezing medium.In some embodiments, the freezing is gradual. In some embodiments,following collection the cells are maintained at room temperature untilfrozen. In some embodiments, the cell-preparation undergoes at least onewashing step in washing medium following cell-collection and prior tofreezing.

As used herein, the terms “obtaining cells” and “cell collection” may beused interchangeably. In some embodiments, the cells of the cellpreparation are frozen within 3-6 hours of collection. In someembodiments, the cell preparation is frozen within up to 6 hours of cellcollection. In some embodiments, the cells of the cell preparations arefrozen within 1, 2, 3, 4, 5, 6, 7, 8 hours of collection. In otherembodiments, the cells of the cell preparations are frozen up to 8, 12,24, 48, 72 hours of collection. In other embodiments, followingcollection the cells are maintained at 2-8° C. until frozen.

In some embodiments, freezing according to the production of an earlyapoptotic cell population comprises: freezing the cell preparation atabout −18° C. to −25° C. followed by freezing the cell preparation atabout −80° C. and finally freezing the cell preparation in liquidnitrogen until thawing. In some embodiments, the freezing according tothe production of an early apoptotic cell population comprises: freezingthe cell preparation at about −18° C. to −25° C. for at least 2 hours,freezing the cell preparation at about −80° C. for at least 2 hours andfinally freezing the cell preparation in liquid nitrogen until thawing.In some embodiments, the cells are kept in liquid nitrogen for at least8, 10 or 12 hours prior to thawing. In some embodiments, the cells ofthe cell preparation are kept in liquid nitrogen until thawing andincubation with apoptosis-inducing incubation medium. In someembodiments, the cells of the cell preparation are kept in liquidnitrogen until the day of hematopoietic stem cell transplantation. Innon-limiting examples, the time from cell collection and freezing topreparation of the final population may be between 1-50 days,alternatively between 6-30 days. In alternative embodiments, the cellpreparation may be kept in liquid nitrogen for longer time periods, suchas at least several months.

In some embodiments, the freezing according to the production of anearly apoptotic cell population comprises freezing the cell preparationat about −18° C. to −25° C. for at least 0.5, 1, 2, 4 hours. In someembodiments, the freezing according to the production of an earlyapoptotic cell population comprises freezing the cell preparation atabout −18° C. to −25° C. for about 2 hours. In some embodiments, thefreezing In the production of an early apoptotic cell populationcomprises freezing the cell preparation at about −80° C. for at least0.5, 1, 2, 4, 12 hours.

In some embodiments, the mononuclear-enriched cell composition mayremain frozen at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 20 months.In some embodiments, the mononuclear-enriched cell composition mayremain frozen at least 0.5, 1, 2, 3, 4, 5 years. In certain embodiments,the mononuclear-enriched cell composition may remain frozen for at least20 months.

In some embodiments, the mononuclear-enriched cell composition is frozenfor at least 8, 10, 12, 18, 24 hours. In certain embodiments, freezingthe mononuclear-enriched cell composition is for a period of at least 8hours. In some embodiments, the mononuclear-enriched cell composition isfrozen for at least about 10 hours. In some embodiments, themononuclear-enriched cell composition is frozen for at least about 12hours. In some embodiments, the mononuclear-enriched cell composition isfrozen for about 12 hours. In some embodiments, the total freezing timeof the mononuclear-enriched cell composition (at about −18° C. to −25°C., at about −80° C. and in liquid nitrogen) is at least 8, 10, 12, 18,24 hours.

In some embodiments, the freezing at least partly induces theearly-apoptotic state in the cells of the mononuclear-enriched cellcomposition. In some embodiments, the freezing medium comprises RPMI1640 medium comprising L-glutamine, Hepes, Hes, dimethyl sulfoxide(DMSO) and plasma. In some embodiments, the plasma in the freezingmedium is an autologous plasma of the donor which donated themononuclear-enriched cells of the population. In some embodiments, thefreezing medium comprises RPMI 1640 medium comprising 2 mM L-glutamine,10 mM Hepes, 5% Hes, 10% dimethyl sulfoxide and 20% v/v plasma.

In some embodiments, the freezing medium comprises an anti-coagulant. Incertain embodiments, at least some of the media used during theproduction of an early apoptotic cell population, including the freezingmedium, the incubation medium and the washing media comprise ananti-coagulant. In certain embodiments, all media used during theproduction of an early apoptotic cell population which comprise ananti-coagulant comprise the same concentration of anti-coagulant. Insome embodiments, anti-coagulant is not added to the final suspensionmedium of the cell population.

In some embodiments, addition of an anti-coagulant at least to thefreezing medium improves the yield of the cell-preparation. In otherembodiments, addition of an anti-coagulant to the freezing mediumimproves the yield of the cell-preparation in the presence of a hightriglyceride level. As used herein, improvement in the yield of thecell-preparation relates to improvement in at least one of: thepercentage of viable cells out of cells frozen, the percentage ofearly-state apoptotic cells out of viable cells and a combinationthereof.

In some embodiments, early apoptotic cells are stable for at least 24hours. In another embodiment, early apoptotic cells are stable for 24hours. In another embodiment, early apoptotic cells are stable for morethan 24 hours. In another embodiment, early apoptotic cells are stablefor at least 36 hours. In another embodiment, early apoptotic cells arestable for 48 hours. In another embodiment, early apoptotic cells arestable for at least 36 hours. In another embodiment, early apoptoticcells are stable for more than 36 hours. In another embodiment, earlyapoptotic cells are stable for at least 48 hours. In another embodiment,early apoptotic cells are stable for 48 hours. In another embodiment,early apoptotic cells are stable for at least 48 hours. In anotherembodiment, early apoptotic cells are stable for more than 48 hours. Inanother embodiment, early apoptotic cells are stable for at least 72hours. In another embodiment, early apoptotic cells are stable for 72hours. In another embodiment, early apoptotic cells are stable for morethan 72 hours.

A skilled artisan would appreciate that the term “stable” encompassesapoptotic cells that remain PS-positive (Phosphatidylserine-positive)with only a very small percent of PI-positive (Propidiumiodide-positive). PI-positive cells provide an indication of membranestability wherein a PI-positive cells permits admission into the cells,showing that the membrane is less stable. In some embodiments, stableearly apoptotic cells remain in early apoptosis for at least 24 hours,for at least 36 hours, for at least 48 hours, or for at least 72 hours.In another embodiment, stable early apoptotic cells remain in earlyapoptosis for 24 hours, for 36 hours, for 48 hours, or for 72 hours. Inanother embodiment, stable early apoptotic cells remain in earlyapoptosis for more than 24 hours, for more than 36 hours, for more than48 hours, or for more than 72 hours. In another embodiment, stable earlyapoptotic cells maintain their state for an extended time period.

In some embodiments, an apoptotic cell population is devoid of cellaggregates. In some embodiments, an apoptotic cell population is devoidof large cell aggregates. In some embodiments, an apoptotic cellpopulation has a reduced number of cell aggregates compared to anapoptotic cell population prepared without adding an anticoagulant in astep other than cell collection (leukapheresis) from the donor. In someembodiments, an apoptotic cell population or a composition thereof,comprises an anticoagulant.

In some embodiments, apoptotic cells are devoid of cell aggregates,wherein said apoptotic cells were obtained from a subject with highblood triglycerides. In some embodiments, blood triglycerides levels ofthe subject are above 150 mg/dL. In some embodiments, an apoptotic cellpopulation is devoid of cell aggregates, wherein said apoptotic cellpopulation is prepared from cells obtained from a subject with normalblood triglycerides. In some embodiments, blood triglycerides levels ofthe subject are equal to or below 150 mg/dL. In some embodiments, cellaggregates produce cell loss during apoptotic cell production methods.

A skilled artisan would appreciate that the terms “aggregates” or “cellaggregates” may encompass the reversible clumping of blood cells underlow shear forces or at stasis. Cell aggregates can be visually observedduring the incubation steps of the production of the apoptotic cells.Cell aggregation can be measured by any method known in the art, forexample by visually imaging samples under a light microscope or usingflow cytometry.

In some embodiments, the anti-coagulant is selected from the groupcomprising: heparin, acid citrate dextrose (ACD) Formula A and acombination thereof. In some embodiments, the anti-coagulant is selectedfrom the group consisting of: heparin, acid citrate dextrose (ACD)Formula A and a combination thereof.

In some embodiments of methods of preparing an early apoptotic cellpopulation and compositions thereof, an anticoagulant is added to atleast one medium used during preparation of the population. In someembodiments, the at least one medium used during preparation of thepopulation is selected from the group consisting of: the freezingmedium, the washing medium, the apoptosis inducing incubation medium,and any combinations thereof.

In some embodiments, the anti-coagulant is selected from the groupconsisting of: Heparin, ACD Formula A and a combination thereof. It isto be noted that other anti-coagulants known in the art may be used,such as, but not limited to Fondaparinaux, Bivalirudin and Argatroban.

In some embodiments, at least one medium used during preparation of thepopulation contains 5% of ACD formula A solution comprising 10 U/mlheparin. In some embodiments, anti-coagulant is not added to the finalsuspension medium of the cell population. As used herein, the terms“final suspension medium” and “administration medium” are usedinterchangeably having all the same qualities and meanings.

In some embodiments, at least one medium used during preparation of thepopulation comprises heparin at a concentration of between 0.1-2.5 U/ml.In some embodiments, at least one medium used during preparation of thepopulation comprises ACD Formula A at a concentration of between 1%-15%v/v. In some embodiments, the freezing medium comprises ananti-coagulant. In some embodiments, the incubation medium comprises ananti-coagulant. In some embodiments, both the freezing medium andincubation medium comprise an anti-coagulant. In some embodiments theanti-coagulant is selected from the group consisting of: heparin, ACDFormula A and a combination thereof.

In some embodiments, the heparin in the freezing medium is at aconcentration of between 0.1-2.5 U/ml. In some embodiments, the ACDFormula A in the freezing medium is at a concentration of between 1%-15%v/v. In some embodiments, the heparin in the incubation medium is at aconcentration of between 0.1-2.5 U/ml. In some embodiments, the ACDFormula A in the incubation medium is at a concentration of between1%-15% v/v. In some embodiments, the anticoagulant is a solution ofacid-citrate-dextrose (ACD) formula A. In some embodiments, theanticoagulant added to at least one medium used during preparation ofthe population is ACD Formula A containing heparin at a concentration of10 U/ml.

In some embodiments, the apoptosis inducing incubation medium used inthe production of an early apoptotic cell population comprises ananti-coagulant. In some embodiments, both the freezing medium andapoptosis inducing incubation medium used in the production of an earlyapoptotic cell population comprise an anti-coagulant. Without wishing tobe bound by any theory or mechanism, in order to maintain a high andstable cell yield in different cell compositions, regardless of the cellcollection protocol, in some embodiments addition of anti-coagulantscomprising adding the anticoagulant to both the freezing medium and theapoptosis inducing incubation medium during production of the apoptoticcell population. In some embodiments, a high and stable cell yieldwithin the composition comprises a cell yield of at least 30%,preferably at least 40%, typically at least 50% cells of the initialpopulation of cells used for induction of apoptosis.

In some embodiments, both the freezing medium and the incubation mediumcomprise an anti-coagulant. In some embodiments, addition of ananti-coagulant both to the incubation medium and freezing medium resultsin a high and stable cell-yield between different preparations of thepopulation regardless of cell-collection conditions, such as, but notlimited to, the timing and/or type of anti-coagulant added during cellcollection. In some embodiments, addition of an anti-coagulant both tothe incubation medium and freezing medium results in a high and stableyield of the cell-preparation regardless of the timing and/or type ofanti-coagulant added during leukapheresis. In some embodiments,production of the cell-preparation in the presence of a hightriglyceride level results in a low and/or unstable cell-yield betweendifferent preparations. In some embodiments, producing thecell-preparation from the blood of a donor having high triglyceridelevel results in a low and/or unstable cell-yield of the cellpreparation. In some embodiments, the term “high triglyceride level”refers to a triglyceride level which is above the normal level of ahealthy subject of the same sex and age. In some embodiments, the term“high triglyceride level” refers to a triglyceride level above about 1.7milimole/liter. As used herein, a high and stable yield refers to a cellyield in the population which is high enough to enable preparation of adose which will demonstrate therapeutic efficiency when administered toa subject. In some embodiments, therapeutic efficiency refers to theability to treat, prevent or ameliorate an immune disease, an autoimmunedisease or an inflammatory disease in a subject. In some embodiments, ahigh and stable cell yield is a cell yield of at least 30%, possibly atleast 40%, typically at least 50% of cells in the population out ofcells initially frozen.

In some embodiments, in case the cell-preparation is obtained from adonor having a high triglyceride level, the donor will take at least onemeasure selected from the group consisting of: takingtriglyceride-lowering medication prior to donation, such as, but notlimited to: statins and/or bezafibrate, fasting for a period of at least8, 10, 12 hours prior to donation, eating an appropriate diet to reduceblood triglyceride level at least 24, 48, 72 hours prior to donating andany combination thereof.

In some embodiments, cell yield in the population relates to cell numberin the composition out of the initial number of cells subjected toapoptosis induction. As used herein, the terms “induction of earlyapoptotic state” and “induction of apoptosis” may be usedinterchangeably.

In some embodiments, the mononuclear-enriched cell composition isincubated in incubation medium following freezing and thawing. In someembodiments, there is at least one washing step between thawing andincubation. As used herein, the terms “incubation medium” and “apoptosisinducing incubation medium” are used interchangeably. In someembodiments, the incubation medium comprises RPMI 1640 mediumsupplemented with L-glutamine, Hepes methylprednisolone and plasma. Insome embodiments, the washing medium comprises 2 mM L-glutamine, 10 mMHepes and 10% v/v blood plasma. In some embodiments, the blood plasma inin the incubation medium is derived from the same donor from whom thecells of the cell preparations are derived. In some embodiments, theblood plasma is added to the incubation medium on the day of incubation.In some embodiments, incubation is performed at 37° C. and 5% CO2.

In some embodiments, the incubation medium comprises methylprednisolone.In some embodiments, the methylprednisolone within the incubation mediumfurther induces the cells in the mononuclear-enriched cell compositionto enter an early-apoptotic state. In some embodiments, the cells in themononuclear-enriched cell composition are induced to enter anearly-apoptotic state both by freezing and incubating in the presence ofmethylprednisolone. In some embodiments, the production of an earlyapoptotic cell population advantageously allows induction of anearly-apoptosis state substantially without induction of necrosis,wherein the cells remain stable at said early-apoptotic state for about24 hours following preparation.

In some embodiments, the incubation medium comprises methylprednisoloneat a concentration of about 10-100 μg/ml. In some embodiments, theincubation medium comprises methylprednisolone at a concentration ofabout 40-60 μg/ml, alternatively about 45-55 μg/ml. In some embodiments,the incubation medium comprises methylprednisolone at a concentration of50 μg/ml.

In some embodiments, the incubation is for about 2-12 hours, possibly4-8 hours, typically for about 5-7 hours. In some embodiments, theincubation is for about 6 hours. In some embodiments, the incubation isfor at least 6 hours. In a preferred embodiment, the incubation is for 6hours.

In some embodiments, the incubation medium comprises an anti-coagulant.In some embodiments, addition of an anti-coagulant to the incubationmedium improves the yield of the cell-preparation. In some embodiments,the anti-coagulant in the incubation medium is of the same concentrationas within the freezing medium. In some embodiments, the incubationmedium comprises an anti-coagulant selected from the group consistingof: heparin, ACD Formula A and a combination thereof. In someembodiments, the anti-coagulant used in the incubation medium is ACDFormula A containing heparin at a concentration of 10 U/ml.

In some embodiments, the incubation medium comprises heparin. In someembodiments, the heparin in the incubation medium is at a concentrationof between 0.1-2.5 U/ml. In some embodiments, the heparin in theincubation medium is at a concentration of between 0.1-2.5 U/ml,possibly between 0.3-0.7 U/ml, typically about 0.5 U/ml. In certainembodiments, the heparin in the incubation medium is at a concentrationof about 0.5 U/ml.

In some embodiments, the incubation medium comprises ACD Formula A. Insome embodiments, the ACD Formula A in the incubation medium is at aconcentration of between 1%-15% v/v. In some embodiments, the ACDFormula A in the incubation medium is at a concentration of between1%-15% v/v, possibly between 4%-7% v/v, typically about 5% v/v. In someembodiments, the ACD Formula A in the incubation medium is at aconcentration of about 5% v/v.

In some embodiments, improvement in the yield of the cell-preparationcomprises improvement in the number of the early-apoptotic viable cellsof the preparation out of the number of frozen cells from which thepreparation was produced.

In some embodiments, addition of an anti-coagulant to the freezingmedium contributes to a high and stable yield between differentpreparations of the pharmaceutical population. In preferableembodiments, addition of an anti-coagulant at least to the freezingmedium and incubation medium results in a high and stable yield betweendifferent preparations of the pharmaceutical composition, regardless tothe cell collection protocol used.

In some embodiments, the freezing medium comprises an anti-coagulantselected from the group consisting of: heparin, ACD Formula A and acombination thereof. In some embodiments, the anti-coagulant used in thefreezing medium is ACD Formula A containing heparin at a concentrationof 10 U/ml. In some embodiments, the freezing medium comprises 5% v/v ofACD Formula A solution comprising heparin at a concentration of 10 U/ml.

In some embodiments, the freezing medium comprises heparin. In someembodiments, the heparin in the freezing medium is at a concentration ofbetween 0.1-2.5 U/ml. In some embodiments, the heparin in the freezingmedium is at a concentration of between 0.1-2.5 U/ml, possibly between0.3-0.7 U/ml, typically about 0.5 U/ml. In certain embodiments, theheparin in the freezing medium is at a concentration of about 0.5 U/ml.

In some embodiments, the freezing medium comprises ACD Formula A. Insome embodiments, the ACD Formula A in the freezing medium is at aconcentration of between 1%-15% v/v. In some embodiments, the ACDFormula A in the freezing medium is at a concentration of between 1%-15%v/v, possibly between 4%-7% v/v, typically about 5% v/v. In someembodiments, the ACD Formula A in the freezing medium is at aconcentration of about 5% v/v.

In some embodiments, addition of an anti-coagulant to the incubationmedium and/or freezing medium results in a high and stable cell yieldwithin the population regardless of the triglyceride level in the bloodof the donor. In some embodiments, addition of an anti-coagulant to theincubation medium and/or freezing medium results in a high and stablecell yield within the composition the invention when obtained from theblood of a donor having normal or high triglyceride level. In someembodiments, addition of an anti-coagulant at least to the incubationmedium, results in a high and stable cell yield within the compositionregardless of the triglyceride level in the blood of the donor. In someembodiments, addition of an anti-coagulant to the freezing medium andincubation medium results in a high and stable cell yield within thecomposition regardless of the triglyceride level in the blood of thedonor.

In some embodiments, the freezing medium and/or incubation medium and/orwashing medium comprise heparin at a concentration of at least 0.1 U/ml,possibly at least 0.3 U/ml, typically at least 0.5 U/ml. In someembodiments, the freezing medium and/or incubation medium and/or washingmedium comprise ACD Formula A at a concentration of at least 1% v/v,possibly at least 3% v/v, typically at least 5% v/v.

In some embodiments, the mononuclear-enriched cell composition undergoesat least one washing step following cell collection and prior to beingre-suspended in the freezing medium and frozen. In some embodiments, themononuclear-enriched cell composition undergoes at least one washingstep following freezing and thawing. In some embodiments, washing stepscomprise centrifugation of the mononuclear-enriched cell compositionfollowed by supernatant extraction and re-suspension in washing medium.

In some embodiments, the mononuclear-enriched cell composition undergoesat least one washing step between each stage of the production of anearly apoptotic cell population. In some embodiments, anti-coagulant isadded to washing media during washing steps throughout the production ofan early apoptotic cell population. In some embodiments, themononuclear-enriched cell composition undergoes at least one washingstep following incubation. In some embodiments, the mononuclear-enrichedcell composition undergoes at least one washing step followingincubation using PBS. In some embodiments, anti-coagulant is not addedto the final washing step prior to re-suspension of the cell-preparationin the administration medium. In some embodiments, anti-coagulant is notadded to the PBS used in the final washing step prior to re-suspensionof the cell-preparation in the administration medium. In certainembodiments, anti-coagulant is not added to the administration medium.

In some embodiments, the cell concentration during incubating is about5×10⁶ cells/ml.

In some embodiments, the mononuclear-enriched cell composition issuspended in an administration medium following freezing, thawing andincubating, thereby resulting in the pharmaceutical population. In someembodiments, the administration medium comprises a suitablephysiological buffer. Non-limiting examples of a suitable physiologicalbuffer are: saline solution, Phoshpate Buffered Saline (PBS), Hank'sBalanced Salt Solution (HBSS), and the like. In some embodiments, theadministration medium comprises PBS. In some embodiments, theadministration medium comprises supplements conducive to maintaining theviability of the cells. In some embodiments, the mononuclear-enrichedcell composition is filtered prior to administration. In someembodiments, the mononuclear-enriched cell composition is filtered priorto administration using a filter of at least 200 μm.

In some embodiments, the mononuclear-enriched cell population isre-suspended in an administration medium such that the final volume ofthe resulting cell-preparation is between 100-1000 ml, possibly between200-800 ml, typically between 300-600 ml.

In some embodiments, cell collection refers to obtaining amononuclear-enriched cell composition. In some embodiments, washingsteps performed during the production of an early apoptotic cellpopulation are performed in a washing medium. In certain embodiments,washing steps performed up until the incubation step of the productionof an early apoptotic cell population are performed in a washing medium.In some embodiments, the washing medium comprises RPMI 1640 mediumsupplemented with L-glutamine and Hepes. In some embodiments, thewashing medium comprises RPMI 1640 medium supplemented with 2 mML-glutamine and 10 mM Hepes.

In some embodiments, the washing medium comprises an anti-coagulant. Insome embodiments, the washing medium comprises an anti-coagulantselected from the group consisting of: heparin, ACD Formula A and acombination thereof. In some embodiments, the concentration of theanti-coagulant in the washing medium is the same concentration as in thefreezing medium. In some embodiments, the concentration of theanti-coagulant in the washing medium is the same concentration as in theincubation medium. In some embodiments, the anti-coagulant used in thewashing medium is ACD Formula A containing heparin at a concentration of10 U/ml.

In some embodiments, the washing medium comprises heparin. In someembodiments, the heparin in the washing medium is at a concentration ofbetween 0.1-2.5 U/ml. In some embodiments, the heparin in the washingmedium is at a concentration of between 0.1-2.5 U/ml, possibly between0.3-0.7 U/ml, typically about 0.5 U/ml. In certain embodiments, theheparin in the washing medium is at a concentration of about 0.5 U/ml.

In some embodiments, the washing medium comprises ACD Formula A. In someembodiments, the ACD Formula A in the washing medium is at aconcentration of between 1%-15% v/v. In some embodiments, the ACDFormula A in the washing medium is at a concentration of between 1%-15%v/v, possibly between 4%-7% v/v, typically about 5% v/v. In someembodiments, the ACD Formula A in the washing medium is at aconcentration of about 5% v/v.

In some embodiments, the mononuclear-enriched cell composition is thawedseveral hours prior to the intended administration of the population toa subject. In some embodiments, the mononuclear-enriched cellcomposition is thawed at about 33° C.-39° C. In some embodiments, themononuclear-enriched cell composition is thawed for about 30-240seconds, preferably 40-180 seconds, most preferably 50-120 seconds.

In some embodiments, the mononuclear-enriched cell composition is thawedat least 10 hours prior to the intended administration of thepopulation, alternatively at least 20, 30, 40 or 50 hours prior to theintended administration of the population. In some embodiments, themononuclear-enriched cell composition is thawed at least 15-24 hoursprior to the intended administration of the population. In someembodiments, the mononuclear-enriched cell composition is thawed atleast about 24 hours prior to the intended administration of thepopulation. In some embodiments, the mononuclear-enriched cellcomposition is thawed at least 20 hours prior to the intendedadministration of the population. In some embodiments, themononuclear-enriched cell composition is thawed 30 hours prior to theintended administration of the population. In some embodiments, themononuclear-enriched cell composition is thawed at least 24 hours priorto the intended administration of the population. In some embodiments,the mononuclear-enriched cell composition undergoes at least one step ofwashing in the washing medium before and/or after thawing.

In some embodiments, the composition further comprisesmethylprednisolone. At some embodiments, the concentration ofmethylprednisolone does not exceed 30 μg/ml.

In some embodiments, the apoptotic cells are used at a high dose. Insome embodiments, the apoptotic cells are used at a high concentration.In some embodiments, human apoptotic polymorphonuclear neutrophils(PMNs) are used. In some embodiments, a group of cells, of which 50% areapoptotic cells, are used. In some embodiments, apoptotic cells areverified by May-Giemsa-stained cytopreps. In some embodiments, viabilityof cells are assessed by trypan blue exclusion. In some embodiments, theapoptotic and necrotic status of the cells are confirmed by annexinV/propidium iodide staining with detection by FACS.

In some embodiments, apoptotic cells disclosed herein comprise nonecrotic cells. In some embodiments, apoptotic cells disclosed hereincomprise less than 1% necrotic cells. In some embodiments, apoptoticcells disclosed herein comprise less than 2% necrotic cells. In someembodiments, apoptotic cells disclosed herein comprise less than 3%necrotic cells. In some embodiments, apoptotic cells disclosed hereincomprise less than 4% necrotic cells. In some embodiments, apoptoticcells disclosed herein comprise less than 5% necrotic cells.

In some embodiments, a dose of about 140×10⁶-210×10⁶ apoptotic cells areadministered. In some embodiments, a dose of about 10-100×10⁶ apoptoticcells is administered. In some embodiments, a dose of about 20×10⁶apoptotic cells is administered. In some embodiments, a dose of about30×10⁶ apoptotic cells is administered. In some embodiments, a dose ofabout 40×10⁶ apoptotic cells is administered. In some embodiments, adose of about 50×10⁶ apoptotic cells is administered. In someembodiments, 60×10⁶ apoptotic cells is administered. In someembodiments, a dose of about 60×10⁶ apoptotic cells is administered. Insome embodiments, a dose of about 70×10⁶ apoptotic cells isadministered. In some embodiments, a dose of about 80×10⁶ apoptoticcells is administered. In some embodiments, a dose of about 90×10⁶apoptotic cells is administered. In some embodiments, a dose of about1-15×10⁷ apoptotic cells is administered. In some embodiments, a dose ofabout 10×10⁷ apoptotic cells is administered. In some embodiments, adose of about 15×10⁷ apoptotic cells is administered.

In some embodiments, a dose of 10×10⁶ apoptotic cells is administered.In another embodiment, a dose of 10×10⁷ apoptotic cells is administered.In another embodiment, a dose of 10×10⁸ apoptotic cells is administered.In another embodiment, a dose of 10×10⁹ apoptotic cells is administered.In another embodiment, a dose of 10×10¹⁰ apoptotic cells isadministered. In another embodiment, a dose of 10×10¹¹ apoptotic cellsis administered. In another embodiment, a dose of 10×10¹² apoptoticcells is administered. In another embodiment, a dose of 10×10⁵ apoptoticcells is administered. In another embodiment, a dose of 10×10⁴ apoptoticcells is administered. In another embodiment, a dose of 10×10³ apoptoticcells is administered. In another embodiment, a dose of 10×10² apoptoticcells is administered.

In some embodiments, a high dose of apoptotic cells is administered. Insome embodiments, a dose of 35×10⁶ apoptotic cells is administered. Inanother embodiment, a dose of 210×10⁶ apoptotic cells is administered.In another embodiment, a dose of 70×10⁶ apoptotic cells is administered.In another embodiment, a dose of 140×10⁶ apoptotic cells isadministered. In another embodiment, a dose of 35-210×10⁶ apoptoticcells is administered.

In some embodiments, a single dose of apoptotic cells is administered.In some embodiments, multiple doses of apoptotic cells are administered.In some embodiments, 2 doses of apoptotic cells are administered. Insome embodiments, 3 doses of apoptotic cells are administered. In someembodiments, 4 doses of apoptotic cells are administered. In someembodiments, 5 doses of apoptotic cells are administered. In someembodiments, 6 doses of apoptotic cells are administered. In someembodiments, 7 doses of apoptotic cells are administered. In someembodiments, 8 doses of apoptotic cells are administered. In someembodiments, 9 doses of apoptotic cells are administered. In someembodiments, more than 9 doses of apoptotic cells are administered. Insome embodiments, multiple doses of apoptotic cells are administered.

In some embodiments, the apoptotic cells may be administered by anymethod known in the art including, but not limited to, intravenous,subcutaneous, intranodal, intratumoral, intrathecal, intrapleural,intraperitoneal and directly to the thymus.

In some embodiments, the apoptotic cells are prepared from cellsobtained from a subject other than the subject that will receive saidapoptotic cells. In some embodiments, the methods as disclosed hereincomprise an additional step that is useful in overcoming rejection ofallogeneic donor cells, including one or more steps described in U.S.Patent Application 20130156794, which is incorporated herein byreference in its entirety. In some embodiments, the methods comprise thestep of full or partial lymphodepletion prior to administration of theapoptotic cells, which in some embodiments, are allogeneic apoptoticcells. In some embodiments, the lymphodepletion is adjusted so that itdelays the host versus graft reaction for a period sufficient to allowthe allogeneic apoptotic cells to control cytokine release. In someembodiments, the methods comprise the step of administering agents thatdelay egression of the allogeneic apoptotic T-cells from lymph nodes,such as 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol (FTY720),5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)pheny-1]1,2,4-oxadiazole(SEW2871), 3-(2-(-hexylphenylamino)-2-oxoethylamino)propanoic acid(W123),2-ammonio-4-(2-chloro-4-(3-phenoxyphenylthio)phenyl)-2-(hydroxymethyl)but-ylhydrogen phosphate (KRP-203 phosphate) or other agents known in the art,may be used as part of the compositions and methods as disclosed hereinto allow the use of allogeneic apoptotic cells having efficacy andlacking initiation of graft vs host disease. In another embodiment, MHCexpression by the allogeneic apoptotic T-cells is silenced to reduce therejection of the allogeneic cells.

In some embodiments, methods comprise producing a population ofmononuclear apoptotic cell comprising a decreased percent ofnon-quiescent non-apoptotic viable cells; a suppressed cellularactivation of any living non-apoptotic cells; or a reduced proliferationof any living non-apoptotic cells; or any combination thereof, saidmethod comprising the following steps, obtaining a mononuclear-enrichedcell population of peripheral blood; freezing said mononuclear-enrichedcell population in a freezing medium comprising an anticoagulant;thawing said mononuclear-enriched cell population; incubating saidmononuclear-enriched cell population in an apoptosis inducing incubationmedium comprising methylprednisolone at a final concentration of about10-100 μg/mL and an anticoagulant; resuspending said apoptotic cellpopulation in an administration medium; and inactivating saidmononuclear-enriched population, wherein said inactivation occursfollowing apoptotic induction, wherein said method produces a populationof mononuclear apoptotic cell comprising a decreased percent ofnon-quiescent non-apoptotic cells; a suppressed cellular activation ofany living non-apoptotic cells; or a reduced proliferation of any livingnon-apoptotic cells; or any combination thereof.

In some embodiments, the methods comprise the step of irradiating apopulation of apoptotic cells derived from a subject prior toadministration of the population of apoptotic cells to the same subject(autologous ApoCells). In some embodiments, the methods comprise thestep of irradiating apoptotic cells derived from a subject prior toadministration of the population of apoptotic cells to a recipient(allogeneic ApoCells).

In some embodiments, cells are irradiated in a way that will decreaseproliferation and/or activation of residual viable cells within theapoptotic cell population. In some embodiments, cells are irradiated ina way that reduces the percent of viable non-apoptotic cells in apopulation. In some embodiments, the percent of viable non-apoptoticcells in an inactivated early apoptotic cell population is reduced toless than 50% of the population. In some embodiments, the percent ofviable non-apoptotic cells in an inactivated early apoptotic cellpopulation is reduced to less than 40% of the population. In someembodiments, the percent of viable non-apoptotic cells in an inactivatedearly apoptotic cell population is reduced to less than 30% of thepopulation. In some embodiments, the percent of viable non-apoptoticcells in an inactivated early apoptotic cell population is reduced toless than 20% of the population. In some embodiments, the percent ofviable non-apoptotic cells in an inactivated early apoptotic cellpopulation is reduced to less than 10% of the population. In someembodiments, the percent of viable non-apoptotic cells in an inactivatedearly apoptotic cell population is reduced to 0% of the population.

In another embodiment, the irradiated apoptotic cells preserve all theirearly apoptotic-, immune modulation-, stability-properties. In anotherembodiment, the irradiation step uses UV radiation. In anotherembodiment, the radiation step uses gamma radiation. In anotherembodiment, the apoptotic cells comprise a decreased percent of livingnon-apoptotic cells, comprise a preparation having a suppressed cellularactivation of any living non-apoptotic cells present within theapoptotic cell preparation, or comprise a preparation having reducedproliferation of any living non-apoptotic cells present within theapoptotic cell preparation, or any combination thereof.

In some embodiments, irradiation of apoptotic cells does not increasethe population of dead cells (PI+) compared with apoptotic cells notirradiated. In some embodiments, irradiation of apoptotic cells does notincrease the population of dead cells (PI+) by more than about 1%compared with apoptotic cells not irradiated. In some embodiments,irradiation of apoptotic cells does not increase the population of deadcells (PI+) by more than about 2% compared with apoptotic cells notirradiated. In some embodiments, irradiation of apoptotic cells does notincrease the population of dead cells (PI+) by more than about 3%compared with apoptotic cells not irradiated. In some embodiments,irradiation of apoptotic cells does not increase the population of deadcells (PI+) by more than about 4% compared with apoptotic cells notirradiated. In some embodiments, irradiation of apoptotic cells does notincrease the population of dead cells (PI+) by more than about 5%compared with apoptotic cells not irradiated. In some embodiments,irradiation of apoptotic cells does not increase the population of deadcells (PI+) by more than about 6% compared with apoptotic cells notirradiated. In some embodiments, irradiation of apoptotic cells does notincrease the population of dead cells (PI+) by more than about 7%compared with apoptotic cells not irradiated. In some embodiments,irradiation of apoptotic cells does not increase the population of deadcells (PI+) by more than about 8% compared with apoptotic cells notirradiated. In some embodiments, irradiation of apoptotic cells does notincrease the population of dead cells (PI+) by more than about 9%compared with apoptotic cells not irradiated. In some embodiments,irradiation of apoptotic cells does not increase the population of deadcells (PI+) by more than about 10% compared with apoptotic cells notirradiated. In some embodiments, irradiation of apoptotic cells does notincrease the population of dead cells (PI+) by more than about 15%compared with apoptotic cells not irradiated. In some embodiments,irradiation of apoptotic cells does not increase the population of deadcells (PI+) by more than about 20%, 25%, 30%, 35%, 40%, 45%, or 50%compared with apoptotic cells not irradiated.

In some embodiments, a cell population comprising a reduced ornon-existent fraction of living non-apoptotic cells may in oneembodiment provide a mononuclear early apoptotic cell population thatdoes not have any living/viable cells. In some embodiments, a cellpopulation comprising a reduced or non-existent fraction of livingnon-apoptotic cells may in one embodiment provide a mononuclearapoptotic cell population that does not elicit GVHD in a recipient.

In some embodiments, use of irradiated ApoCells removes the possiblegraft versus leukemia effect use of an apoptotic population (thatincludes a minor portion of viable cells) may cause, demonstrating thatthe effects shown here in the Examples (See Example 8) result from theapoptotic cells and not from a viable proliferating population of cellswith cellular activity, present within the apoptotic cell population.

In another embodiment, the methods comprise the step of irradiatingapoptotic cells derived from WBCs from a donor prior to administrationto a recipient. In some embodiments, cells are irradiated in a way thatwill avoid proliferation and/or activation of residual viable cellswithin the apoptotic cell population. In another embodiment, theirradiated apoptotic cells preserve all their early apoptotic-, immunemodulation-, stability-properties. In another embodiment, theirradiation step uses UV radiation. In another embodiment, the radiationstep uses gamma radiation. In another embodiment, the apoptotic cellscomprise a decreased percent of living non-apoptotic cells, comprise apreparation having a suppressed cellular activation of any livingnon-apoptotic cells present within the apoptotic cell preparation, orcomprise a preparation having reduced proliferation of any livingnon-apoptotic cells present within the apoptotic cell preparation, orany combination thereof.

In some embodiments, apoptotic cells comprise a pooled mononuclearapoptotic cell preparation. In some embodiments, a pooled mononuclearapoptotic cell preparation comprises mononuclear cells in an earlyapoptotic state, wherein said pooled mononuclear apoptotic cellscomprise a decreased percent of living non-apoptotic cells, apreparation having a suppressed cellular activation of any livingnon-apoptotic cells, or a preparation having reduced proliferation ofany living non-apoptotic cells, or any combination thereof. In anotherembodiment, the pooled mononuclear apoptotic cells have been irradiated.In another embodiment, disclosed herein is a pooled mononuclearapoptotic cell preparation that in some embodiments, originates from thewhite blood cell fraction (WBC) obtained from donated blood.

In some embodiments, the apoptotic cell preparation is irradiated. Inanother embodiment, said irradiation comprises gamma irradiation or UVirradiation. In yet another embodiment, the irradiated preparation has areduced number of non-apoptotic cells compared with a non-irradiatedapoptotic cell preparation. In another embodiment, the irradiatedpreparation has a reduced number of proliferating cells compared with anon-irradiated apoptotic cell preparation. In another embodiment, theirradiated preparation has a reduced number of potentiallyimmunologically active cells compared with a non-irradiated apoptoticcell population.

In some embodiments, pooled blood comprises 3rd party blood not matchedbetween donor and recipient.

A skilled artisan would appreciate that the term “pooled” may encompassblood collected from multiple donors, prepared and possibly stored forlater use. This combined pool of blood may then be processed to producea pooled mononuclear apoptotic cell preparation. In another embodiment,a pooled mononuclear apoptotic cell preparation ensures that a readilyavailable supply of mononuclear apoptotic cells is available. In anotherembodiment, cells are pooled just prior to the incubation step whereinapoptosis is induced. In another embodiment, cells are pooled followingthe incubation step at the step of resuspension. In another embodiment,cells are pooled just prior to an irradiation step. In anotherembodiment, cells are pooled following an irradiation step. In anotherembodiment, cells are pooled at any step in the methods of preparation.

In some embodiments, a pooled apoptotic cell preparation is derived fromcells present in between about 2 and 25 units of blood. In anotherembodiment, said pooled apoptotic cell preparation is comprised of cellspresent in between about 2-5, 2-10, 2-15, 2-20, 5-10, 5-15, 5-20, 5-25,10-15, 10-20, 10-25, 6-13, or 6-25 units of blood. In anotherembodiment, said pooled apoptotic cell preparation is comprised of cellspresent in about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24 or 25 units of blood. The number of units ofblood needed is also dependent upon the efficiency of WBC recovery fromblood. For example, low efficiency WBC recovery would lead to the needfor additional units, while high efficiency WBC recovery would lead tofewer units needed. In some embodiments, each unit is a bag of blood. Inanother embodiment, a pooled apoptotic cell preparation is comprised ofcells present in at least 25 units of blood, at least 50 units of blood,or at least 100 units of blood.

In some embodiments, the units of blood comprise white blood cell (WBC)fractions from blood donations. In another embodiment, the donations maybe from a blood center or blood bank. In another embodiment, thedonations may be from donors in a hospital gathered at the time ofpreparation of the pooled apoptotic cell preparation. In anotherembodiment, units of blood comprising WBCs from multiple donors aresaved and maintained in an independent blood bank created for thepurpose of compositions and methods thereof as disclosed herein. Inanother embodiment, a blood bank developed for the purpose ofcompositions and methods thereof as disclosed herein, is able to supplyunits of blood comprising WBC from multiple donors and comprises aleukapheresis unit.

In some embodiments, the units of pooled WBCs are not restricted by HLAmatching. Therefore, the resultant pooled apoptotic cell preparationcomprises cell populations not restricted by HLA matching. Accordingly,in certain embodiments a pooled mononuclear apoptotic cell preparationcomprises allogeneic cells.

An advantage of a pooled mononuclear apoptotic cell preparation that isderived from pooled WBCs not restricted by HLA matching, is a readilyavailable source of WBCs and reduced costs of obtaining WBCs.

In some embodiments, pooled blood comprises blood from multiple donorsindependent of HLA matching. In another embodiment, pooled bloodcomprises blood from multiple donors wherein HLA matching with therecipient has been taken into consideration. For example, wherein 1 HLAallele, 2 HLA alleles, 3 HLA alleles, 4 HLA alleles, 5 HLA alleles, 6HLA alleles, or 7 HLA alleles have been matched between donors andrecipient. In another embodiment, multiple donors are partially matched,for example some of the donors have been HLA matched wherein 1 HLAallele, 2 HLA alleles, 3 HLA alleles, 4 HLA alleles, 5 HLA alleles, 6HLA alleles, or 7 HLA alleles have been matched between some of thedonors and recipient. Each possibility comprises an embodiment asdisclosed herein.

In certain embodiments, some viable non-apoptotic cells (apoptosisresistant) may remain following the induction of apoptosis stepdescribed below (Example 1). The presence of these viable non-apoptoticcells is, in some embodiments, is observed prior to an irradiation step.These viable non-apoptotic cells may be able to proliferate or beactivated. In some embodiments, the pooled mononuclear apoptotic cellpreparation derived from multiple donors may be activated against thehost, activated against one another, or both.

In some embodiments, an irradiated cell preparation as disclosed hereinhas suppressed cellular activation and reduced proliferation comparedwith a non-irradiated cell preparation. In another embodiment, theirradiation comprises gamma irradiation or UV irradiation. In anotherembodiment, an irradiated cell preparation has a reduced number ofnon-apoptotic cells compared with a non-irradiated cell preparation. Inanother embodiment, the irradiation comprises about 15 Grey units (Gy).In another embodiment, the irradiation comprises about 20 Grey units(Gy). In another embodiment, the irradiation comprises about 25 Greyunits (Gy). In another embodiment, the irradiation comprises about 30Grey units (Gy). In another embodiment, the irradiation comprises about35 Grey units (Gy). In another embodiment, the irradiation comprisesabout 40 Grey units (Gy). In another embodiment, the irradiationcomprises about 45 Grey units (Gy). In another embodiment, theirradiation comprises about 50 Grey units (Gy). In another embodiment,the irradiation comprises about 55 Grey units (Gy). In anotherembodiment, the irradiation comprises about 60 Grey units (Gy). Inanother embodiment, the irradiation comprises about 65 Grey units (Gy).In another embodiment, the irradiation comprises up to 2500 Gy. Inanother embodiment, an irradiated pooled apoptotic cell preparationmaintains the same or a similar apoptotic profile, stability andefficacy as a non-irradiated pooled apoptotic cell preparation.

In some embodiments, a pooled mononuclear apoptotic cell preparation asdisclosed herein is stable for up to 24 hours. In another embodiment, apooled mononuclear apoptotic cell preparation is stable for at least 24hours. In another embodiment, a pooled mononuclear apoptotic cellpreparation is stable for more than 24 hours. In yet another embodiment,a pooled mononuclear apoptotic cell preparation as disclosed herein isstable for up to 36 hours. In still another embodiment, a pooledmononuclear apoptotic cell preparation is stable for at least 36 hours.In a further embodiment, a pooled mononuclear apoptotic cell preparationis stable for more than 36 hours. In another embodiment, a pooledmononuclear apoptotic cell preparation as disclosed herein is stable forup to 48 hours. In another embodiment, a pooled mononuclear apoptoticcell preparation is stable for at least 48 hours. In another embodiment,a pooled mononuclear apoptotic cell preparation is stable for more than48 hours.

In some embodiments, methods of producing the pooled cell preparationcomprising an irradiation step preserves the early apoptotic, immunemodulation, and stability properties observed in an apoptoticpreparation derived from a single match donor wherein the cellpreparation may not include an irradiation step. In another embodiment,a pooled mononuclear apoptotic cell preparation as disclosed herein doesnot elicit a graft versus host disease (GVHD) response.

Irradiation of the cell preparation is considered safe in the art.Irradiation procedures are currently performed on a routine basis todonated blood to prevent reactions to WBC.

In another embodiment, the percent of apoptotic cells in a pooledmononuclear apoptotic cell preparation as disclosed herein is close to100%, thereby reducing the fraction of living non-apoptotic cells in thecell preparation. In some embodiments, the percent of apoptotic cells isat least 40%. In another embodiment, the percent of apoptotic cells isat least 50%. In yet another embodiment, the percent of apoptotic cellsis at least 60%. In still another embodiment, the percent of apoptoticcells is at least 70%. In a further embodiment, the percent of apoptoticcells is at least 80%. In another embodiment, the percent of apoptoticcells is at least 90%. In yet another embodiment, the percent ofapoptotic cells is at least 99%. Accordingly, a cell preparationcomprising a reduced or non-existent fraction of living non-apoptoticcells may in one embodiment provide a pooled mononuclear apoptotic cellpreparation that does not elicit GVHD in a recipient. Each possibilityrepresents an embodiment as disclosed herein.

Alternatively, in another embodiment, the percentage of livingnon-apoptotic WBC is reduced by specifically removing the living cellpopulation, for example by targeted precipitation. In anotherembodiment, the percent of living non-apoptotic cells may be reducedusing magnetic beads that bind to phosphatidylserine. In anotherembodiment, the percent of living non-apoptotic cells may be reducedusing magnetic beads that bind a marker on the cell surface ofnon-apoptotic cells but not apoptotic cells. In another embodiment, theapoptotic cells may be selected for further preparation using magneticbeads that bind to a marker on the cell surface of apoptotic cells butnot non-apoptotic cells. In yet another embodiment, the percentage ofliving non-apoptotic WBC is reduced by the use of ultrasound.

In one embodiment the apoptotic cells are from pooled third partydonors.

In some embodiments, a pooled cell preparation comprises at least onecell type selected from the group consisting of: lymphocytes, monocytesand natural killer cells. In another embodiment, a pooled cellpreparation comprises an enriched population of mononuclear cells. Insome embodiments, a pooled mononuclear is a mononuclear enriched cellpreparation comprises cell types selected from the group consisting of:lymphocytes, monocytes and natural killer cells. In another embodiment,the mononuclear enriched cell preparation comprises no more than 15%,alternatively no more than 10%, typically no more than 5%polymorphonuclear leukocytes, also known as granulocytes (i.e.,neutrophils, basophils and eosinophils). In another embodiment, a pooledmononuclear cell preparation is devoid of granulocytes.

In another embodiment, the pooled mononuclear enriched cell preparationcomprises no more than 15%, alternatively no more than 10%, typically nomore than 5% CD15^(high) expressing cells. In some embodiments, a pooledapoptotic cell preparation comprises less than 15% CD15 high expressingcells.

In some embodiments, the pooled mononuclear enriched cell preparationdisclosed herein comprises at least 80% mononuclear cells, at least 85%mononuclear cells, alternatively at least 90% mononuclear cells, or atleast 95% mononuclear cells, wherein each possibility is a separateembodiment disclosed herein. According to some embodiments, the pooledmononuclear enriched cell preparation disclosed herein comprises atleast 85% mononuclear cells.

In another embodiment, any pooled cell preparation that has a finalpooled percent of mononuclear cells of at least 80% is considered apooled mononuclear enriched cell preparation as disclosed herein. Thus,pooling cell preparations having increased polymorphonuclear cells (PMN)with cell preparations having high mononuclear cells with a resultant“pool” of at least 80% mononuclear cells comprises a preparation asdisclosed herein. According to some embodiments, mononuclear cellscomprise lymphocytes and monocytes.

A skilled artisan would appreciate that the term “mononuclear cells” mayencompass leukocytes having a one lobed nucleus. In another embodiment,a pooled apoptotic cell preparation as disclosed herein comprises lessthan 5% polymorphonuclear leukocytes.

In some embodiments, the apoptotic cells are T-cells. In anotherembodiment, the apoptotic cells are derived from the same pooled thirdparty donor T-cells as the CAR T-cells. In another embodiment, theapoptotic cells are derived from the CAR T-cell population.

Surprisingly, the apoptotic cells reduce production of cytokinesassociated with the cytokine storm including but not limited to IL-6,and interferon-gamma (IFN-γ), alone or in combination, while theeffectiveness of CAR T-cell therapy was maintained (Example 2). In oneembodiment, the apoptotic cells affect cytokine expression levels inmacrophages. In another embodiment, the apoptotic cells reduce cytokineexpression levels in macrophages. In one embodiment, the apoptotic cellssuppress cytokine expression levels in macrophages. In one embodiment,the apoptotic cells inhibit cytokine expression levels in macrophages.In one embodiment, the apoptotic cells maintain IFN-γ levels to match ornearly match levels present prior to CAR-T cell administration. Inanother embodiment, apoptotic cells affect cytokine expression levels inmacrophages but do not affect cytokine expression levels in the CART-cells. In another embodiment, the apoptotic cells affect cytokineexpression levels in DCs, but do not affect cytokine expression levelsin the CAR T-cells. It was therefore unexpected that apoptotic cellswould be useful in maintaining the effectiveness CAR T-cell therapy.

In another embodiment, the effect of apoptotic cells on cytokineexpression levels in macrophages, DCs, or a combination thereof, resultsin reduction of CRS. In another embodiment, the effect of apoptoticcells on cytokine expression levels in macrophages, DCs, or acombination thereof, results in reduction of severe CRS. In anotherembodiment, the effect of apoptotic cells on cytokine expression levelsin macrophages, DCs, or a combination thereof, results in suppression ofCRS. In another embodiment, the effect of apoptotic cells on cytokineexpression levels in macrophages, DCs, or a combination thereof, resultsin suppression of severe CRS. In another embodiment, the effect ofapoptotic cells on cytokine expression levels in macrophages, DCs, or acombination thereof, results in inhibition of CRS. In anotherembodiment, the effect of apoptotic cells on cytokine expression levelsin macrophages, DCs, or a combination thereof, results in inhibition ofsevere CRS. In another embodiment, the effect of apoptotic cells oncytokine expression levels in macrophages, DCs, or a combinationthereof, results in prevention of CRS. In another embodiment, the effectof apoptotic cells on cytokine expression levels in macrophages, DCs, ora combination thereof, results in prevention of severe CRS.

In another embodiment, the apoptotic cells trigger death of T-cells, butnot via changes in cytokine expression levels.

In another embodiment, apoptotic cells antagonize the priming ofmacrophages and dendritic cells to secrete cytokines that wouldotherwise amplify the cytokine storm. In another embodiment, apoptoticcells increase Tregs which suppress the inflammatory response and/orprevent excess release of cytokines.

In some embodiments, administration of apoptotic cells inhibits one ormore pro-inflammatory cytokines. In some embodiments, thepro-inflammatory cytokine comprises IL-beta, IL-6, TNF-alpha, orIFN-gamma, or any combination thereof. In some embodiments, inhibitionof one or more pro-inflammatory cytokines comprises downregulation ofpr0-inflammatory cytokines, wherein a reduced amount of one or morepro-inflammatory cytokines is secreted.

In another embodiment, administration of apoptotic cells promotes thesecretion of one or more anti-inflammatory cytokines. In someembodiments, the anti-inflammatory cytokine comprises TGF-beta, IL10, orPGE2, or any combination thereof.

In some embodiments, administration of apoptotic cells inhibits one ormore pro-inflammatory cytokine and inhibits on or more anti-inflammatorycytokine. In some embodiments, inhibition of one or morepro-inflammatory cytokine and one or more anti-inflammatory cytokinecomprises downregulation of the one or more pro-inflammatory cytokinesfollowed by downregulation of one or more anti-inflammatory cytokine,wherein a reduced amount of the one or more pro-inflammatory cytokinesand the one or move anti-inflammatory cytokine is secreted. A skilledartisan would appreciate that apoptotic cells may therefore have abeneficial effect on aberrant innate immune response, withdownregulation of both anti- and pro-inflammatory cytokines. In someembodiments, this beneficial effect may follow recognition of PAMPs andDAMPs by components of the innate immune system.

In another embodiment, administration of apoptotic cells inhibitsdendritic cell maturation following exposure to TLR ligands. In anotherembodiment, administration of apoptotic cells creates potentiallytolerogenic dendritic cells, which in some embodiments, are capable ofmigration, and in some embodiments, the migration is due to CCR7. Inanother embodiment, administration of apoptotic cells elicits varioussignaling events which in one embodiment is TAM receptor signaling(Tyro3, Axl and Mer) which in some embodiments, inhibits inflammation inantigen-presenting cells.

In some embodiments, Tyro-3, Axl, and Mer constitute the TAM family ofreceptor tyrosine kinases (RTKs) characterized by a conserved sequencewithin the kinase domain and adhesion molecule-like extracellulardomains. In another embodiment, administration of apoptotic cellsactivates signaling through MerTK. In another embodiment, administrationof apoptotic cells activates the phosphatidylinositol 3-kinase(PI3K)/AKT pathway, which in some embodiments, negatively regulatesNF-κB. In another embodiment, administration of apoptotic cellsnegatively regulates the inflammasome which in one embodiment leads toinhibition of pro-inflammatory cytokine secretion, DC maturation, or acombination thereof. In another embodiment, administration of apoptoticcells upregulates expression of anti-inflammatory genes such as Nr4a,Thbs1, or a combination thereof. In another embodiment, administrationof apoptotic cells induces a high level of AMP which in someembodiments, is accumulated in a Pannexin1-dependent manner. In anotherembodiment, administration of apoptotic cells suppresses inflammation.

In some embodiments, methods of use of early apoptotic cells, asdescribed herein, includes use of the early apoptotic cells or acomposition thereof, in combination with an antibody. In someembodiments, the antibody is directed against a tumor cell antigen. Inanother embodiment, the antibody is directed against CD20. In anotherembodiment, the antibody is rituximab (Rtx).

In some embodiments, early apoptotic cells and an antibody are comprisedin the same composition. In some embodiments, early apoptotic cells andan antibody are comprised in different compositions. In someembodiments, administration of a combination of early apoptotic cellsand an antibody, or composition(s) thereof are concurrent. In someembodiments, administration of a combination of early apoptotic cellsand an antibody, or composition(s) thereof comprises administration ofapoptotic cells or a composition thereof, prior to the antibody. In someembodiments, administration of a combination of early apoptotic cellsand an antibody, or composition(s) thereof comprises administration ofapoptotic cells or a composition thereof, following administration ofthe antibody.

In another embodiment, the antibody is Trastuzumab (Herceptin;Genentech): humanized IgG1, which is directed against ERBB2. In anotherembodiment, the antibody is Bevacizumab (Avastin; Genentech/Roche):humanized IgG1, which is directed against VEGF. In another embodiment,the antibody is Cetuximab (Erbitux; Bristol-Myers Squibb): chimerichuman-murine IgG1, which is directed against EGFR. In anotherembodiment, the antibody is Panitumumab (Vectibix; Amgen): human IgG2,which is directed against EGFR. In another embodiment, the antibody isIpilimumab (Yervoy; Bristol-Myers Squibb): IgG1, which is directedagainst CTLA4.

In another embodiment, the antibody is Alemtuzumab (Campath; Genzyme):humanized IgG1, which is directed against CD52. In another embodiment,the antibody is Ofatumumab (Arzerra; Genmab): human IgG1, which isdirected against CD20. In another embodiment, the antibody is Gemtuzumabozogamicin (Mylotarg; Wyeth): humanized IgG4, which is directed againstCD33. In another embodiment, the antibody is Brentuximab vedotin(Adcetris; Seattle Genetics): chimeric IgG1, which is directed againstCD30. In another embodiment, the antibody is 90Y-labelled ibritumomabtiuxetan (Zevalin; IDEC Pharmaceuticals): murine IgG1, which is directedagainst CD20. In another embodiment, the antibody is 131I-labelledtositumomab (Bexxar; GlaxoSmithKline): murine IgG2, which is directedagainst CD20.

In another embodiment, the antibody is Ramucirumab, which is directedagainst vascular endothelial growth factor receptor-2 (VEGFR-2). Inanother embodiment, the antibody is ramucirumab (Cyramza Injection, EliLilly and Company), blinatumomab (BLINCYTO, Amgen Inc.), pembrolizumab(KEYTRUDA, Merck Sharp & Dohme Corp.), obinutuzumab (GAZYVA, Genentech,Inc.; previously known as GA101), pertuzumab injection (PERJETA,Genentech, Inc.), or denosumab (Xgeva, Amgen Inc.). In anotherembodiment, the antibody is Basiliximab (Simulect; Novartis). In anotherembodiment, the antibody is Daclizumab (Zenapax; Roche).

In another embodiment, the antibody administered in combination withapoptotic cells is directed to a tumor or cancer antigen or a fragmentthereof, that is described herein and/or that is known in the art. Inanother embodiment, the antibody is directed to a tumor-associatedantigen. In another embodiment, the antibody is directed to atumor-associated antigen or a fragment thereof that is an angiogenicfactor.

In some embodiments, antibodies described herein may be used incombination with compositions described herein, for example but notlimited to a composition comprising early apoptotic cells.

Apoptotic Cell Supernatants (ApoSup and ApoSup Mon)

In some embodiments, compositions for use in the methods and treatmentsas disclosed herein include an apoptotic cell supernatant as disclosedherein.

In some embodiments, the apoptotic cell supernatant is obtained by amethod comprising the steps of a) providing apoptotic cells, b)culturing the apoptotic cells of step a), and c) separating thesupernatant from the cells.

In some embodiments, apoptotic cells for use making an apoptotic cellsupernatant as disclosed herein are autologous with a subject undergoingtherapy. In another embodiment, apoptotic cells for use in making anapoptotic cell supernatant disclosed herein are allogeneic with asubject undergoing therapy.

The “apoptotic cells” from which the apoptotic cell supernatant isobtained may be cells chosen from any cell type of a subject, or anycommercially available cell line, subjected to a method of inducingapoptosis known to the person skilled in the art. The method of inducingapoptosis may be hypoxia, ozone, heat, radiation, chemicals, osmoticpressure, pH shift, X-ray irradiation, gamma-ray irradiation, UVirradiation, serum deprivation, corticoids or combinations thereof, orany other method described herein or known in the art. In anotherembodiment, the method of inducing apoptosis produces apoptotic cells inan early apoptotic state.

In some embodiments, the apoptotic cells are leukocytes.

In an embodiment, said apoptotic leukocytes are derived from peripheralblood mononuclear cells (PBMC). In another embodiment, said leukocytesare from pooled third party donors. In another embodiment, saidleukocytes are allogeneic.

According to some embodiments, the apoptotic cells are provided byselecting non-adherent leukocytes and submitting them to apoptosisinduction, followed by a cell culture step in culture medium.“Leukocytes” used to make the apoptotic cell-phagocyte supernatant maybe derived from any lineage, or sub-lineage, of nucleated cells of theimmune system and/or hematopoietic system, including but not limited todendritic cells, macrophages, masT-cells, basophils, hematopoietic stemcells, bone marrow cells, natural killer cells, and the like. Theleukocytes may be derived or obtained in any of various suitable ways,from any of various suitable anatomical compartments, according to anyof various commonly practiced methods, depending on the application andpurpose, desired leukocyte lineage, etc. In some embodiments, the sourceleukocytes are primary leukocytes. In another embodiment, the sourceleukocytes are primary peripheral blood leukocytes.

Primary lymphocytes and monocytes may be conveniently derived fromperipheral blood. Peripheral blood leukocytes include 70-95 percentlymphocytes, and 5-25 percent monocytes.

Methods for obtaining specific types of source leukocytes from blood areroutinely practiced. Obtaining source lymphocytes and/or monocytes canbe achieved, for example, by harvesting blood in the presence of ananticoagulant, such as heparin or citrate. The harvested blood is thencentrifuged over a Ficoll cushion to isolate lymphocytes and monocytesat the gradient interface, and neutrophils and erythrocytes in thepellet.

Leukocytes may be separated from each other via standard immunomagneticselection or immunofluorescent flow cytometry techniques according totheir specific surface markers, or via centrifugal elutriation. Forexample, monocytes can be selected as the CD14+ fraction, T-lymphocytescan be selected as CD3+ fraction, B-lymphocytes can be selected as theCD19+ fraction, macrophages as the CD206+ fraction.

Lymphocytes and monocytes may be isolated from each other by subjectingthese cells to substrate-adherent conditions, such as by static culturein a tissue culture-treated culturing recipient, which results inselective adherence of the monocytes, but not of the lymphocytes, to thecell-adherent substrate.

Leukocytes may also be obtained from peripheral blood mononuclear cells(PBMCs), which may be isolated as described herein.

One of ordinary skill in the art will possess the necessary expertise tosuitably culture primary leukocytes so as to generate desired quantitiesof cultured source leukocytes as disclosed herein, and ample guidancefor practicing such culturing methods is available in the literature ofthe art.

One of ordinary skill in the art will further possess the necessaryexpertise to establish, purchase, or otherwise obtain suitableestablished leukocyte cell lines from which to derive the apoptoticleukocytes. Suitable leukocyte cell lines may be obtained fromcommercial suppliers, such as the American Tissue Type Collection(ATCC). It will be evident to the person skilled in the art that sourceleukocytes should not be obtained via a technique which willsignificantly interfere with their capacity to produce the apoptoticleukocytes.

In another embodiment, the apoptotic cells may be apoptotic lymphocytes.Apoptosis of lymphocytes, such as primary lymphocytes, may be induced bytreating the primary lymphocytes with serum deprivation, acorticosteroid, or irradiation. In another embodiment, inducingapoptosis of primary lymphocytes via treatment with a corticosteroid iseffected by treating the primary lymphocytes with dexamethasone. Inanother embodiment, with dexamethasone at a concentration of about 1micromolar. In another embodiment, inducing apoptosis of primarylymphocytes via irradiation is effected by treating the primarylymphocytes with gamma-irradiation. In another embodiment, with a dosageof about 66 rad. Such treatment results in the generation of apoptoticlymphocytes suitable for the co-culture step with phagocytes.

In a further embodiment, apoptotic cells may be apoptotic monocytes,such as primary monocytes. To generate apoptotic monocytes the monocytesare subjected to in vitro conditions of substrate/surface-adherenceunder conditions of serum deprivation. Such treatment results in thegeneration of non-pro-inflammatory apoptotic monocytes suitable for theco-culture step with phagocytes.

In other embodiments, the apoptotic cells may be any apoptotic cellsdescribed herein, including allogeneic apoptotic cells, third partyapoptotic cells, and pools of apoptotic cells.

In other embodiments, the apoptotic cell supernatant may be obtainedthrough the co-culture of apoptotic cells with other cells.

Thus, in some embodiments, the apoptotic cell supernatant is anapoptotic cell supernatant obtained by a method comprising the steps ofa) providing apoptotic cells, b) providing other cells, c) optionallywashing the cells from step a) and b), d) co-culturing the cells of stepa) and b), and optionally e) separating the supernatant from the cells.

In some embodiments, the other cells co-cultured with the apoptoticcells are white blood cells.

Thus, in some embodiments, the apoptotic cell supernatant is anapoptotic cell-white blood cell supernatant obtained by a methodcomprising the steps of a) providing apoptotic cells, b) providing whiteblood cells, c) optionally washing the cells from step a) and b), d)co-culturing the cells of step a) and b), and optionally e) separatingthe supernatant from the cells.

In some embodiments, the white blood cells may be phagocytes, such asmacrophages, monocytes or dendritic cells.

In some embodiments, the white blood cells may be B cells, T-cells, ornatural killer (NK cells).

Thus, in some embodiments, compositions for use in the methods andtreatments as disclosed herein include apoptotic cell-phagocytesupernatants as described in WO 2014/106666, which is incorporated byreference herein in its entirety. In another embodiment, apoptoticcell-phagocyte supernatants for use in compositions and methods asdisclosed herein are produced in any way that is known in the art.

In some embodiments, the apoptotic cell-phagocyte supernatant isobtained from a co-culture of phagocytes with apoptotic cells,

In some embodiments, the apoptotic cell-phagocyte supernatant isobtained by a method comprising the steps of a) providing phagocytes, b)providing apoptotic cells, c) optionally washing the cells from step a)and b), d) co-culturing the cells of step a) and b), and optionally e)separating the supernatant from the cells.

The term “phagocytes” denotes cells that protect the body by ingesting(phagocytosing) harmful foreign particles, bacteria, and dead or dyingcells. Phagocytes include for example cells called neutrophils,monocytes, macrophages, dendritic cells, and mast T-cells,preferentially dendritic cells and monocytes/macrophages. The phagocytesmay be dendritic cells (CD4+ HLA-DR+ Lineage-BDCA1/BDCA3+), macrophages(CD14+ CD206+ HLA-DR+), or derived from monocytes (CD14+). Techniques todistinguish these different phagocytes are known to the person skilledin the art.

In an embodiment, monocytes are obtained by a plastic adherence step.Said monocytes can be distinguished from B and T-cells with the markerCD14+, whereas unwanted B cells express CD19+ and T-cells CD3+. AfterMacrophage Colony Stimulating Factor (M-CSF) induced maturation theobtained macrophages are in some embodiments, positive for the markersCD14+, CD206+, HLA-DR+.

In an embodiment, said phagocytes are derived from peripheral bloodmononuclear cells (PBMC).

Phagocytes may be provided by any method known in the art for obtainingphagocytes. In some embodiments, phagocytes such as macrophages ordendritic cells can be directly isolated from a subject or be derivedfrom precursor cells by a maturation step.

In some embodiments, macrophages may be directly isolated from theperitoneum cavity of a subject and cultured in complete RRPMI medium.Macrophages can also be isolated from the spleen.

Phagocytes are also obtainable from peripheral blood monocytes. In saidexample, monocytes when cultured differentiate into monocyte-derivedmacrophages upon addition of, without limitation to, macrophage colonystimulating factor (M-CSF) to the cell culture media.

For example, phagocytes may be derived from peripheral blood mononuclearcells (PBMC). For example, PBMC may be isolated from cytapheresis bagfrom an individual through Ficoll gradient centrifugation, plated in acell-adherence step for 90 min in complete RPMI culture medium (10% FBS,1% Penicillin/Streptomycin). Non-adherent T-cells are removed by aplastic adherence step, and adherent T-cells cultured in complete RPMImilieu supplemented with recombinant human M-CSF. After the cultureperiod, monocyte-derived macrophages are obtained.

Phagocytes can be selected by a cell-adherence step. Said “celladherence step” means that phagocytes or cells which can mature intophagocytes are selected via culturing conditions allowing the adhesionof the cultured cells to a surface, a cell adherent surface (e.g. atissue culture dish, a matrix, a sac or bag with the appropriate type ofnylon or plastic). A skilled artisan would appreciate that the term“Cell adherent surfaces” may encompass hydrophilic and negativelycharged, and may be obtained in any of various ways known in the art, Inanother embodiment by modifying a polystyrene surface using, forexample, corona discharge, or gas-plasma. These processes generatehighly energetic oxygen ions which graft onto the surface polystyrenechains so that the surface becomes hydrophilic and negatively charged.Culture recipients designed for facilitating cell-adherence thereto areavailable from various commercial suppliers (e.g. Corning, Perkin-Elmer,Fisher Scientific, Evergreen Scientific, Nunc, etc.).

B cells, T-cells and NK cells may be provided by any method known in theart for obtaining such cells. In some embodiments, B cells, T-cells orNK cells can be directly isolated from a subject or be derived fromprecursor cells by a maturation step. In another embodiment, the B, T orNK cells can be from a B, T or NK cell line. One of ordinary skill inthe art will possess the necessary expertise to establish, purchase, orotherwise obtain suitable established B cells, T-cells and NK celllines. Suitable cell lines may be obtained from commercial suppliers,such as the American Tissue Type Collection (ATCC).

In an embodiment, said apoptotic cells and said white blood cells, suchas the phagocytes, B, T or NK cells, are cultured individually prior tothe co-culture step d).

The cell maturation of phagocytes takes place during cell culture, forexample due to addition of maturation factors to the media. In oneembodiment said maturation factor is M-CSF, which may be used forexample to obtain monocyte-derived macrophages.

The culture step used for maturation or selection of phagocytes mighttake several hours to several days. In another embodiment saidpre-mature phagocytes are cultured for 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58 hours in an appropriate culture medium.

The culture medium for phagocytes is known to the person skilled in theart and can be for example, without limitation, RPMI, DMEM, X-vivo andUltraculture milieus.

In an embodiment, co-culture of apoptotic cells and phagocytes takesplace in a physiological solution.

Prior to this “co-culture”, the cells may be submitted to a washingstep. In some embodiments, the white blood cells (e.g. the phagocytes)and the apoptotic cells are washed before the co-culture step. Inanother embodiment, the cells are washed with PBS.

During said co-culture the white blood cells (e.g. the phagocytes suchas macrophages, monocytes, or phagocytes, or the B, T or NK cells) andthe apoptotic cells may be mixed in a ratio of 10:1, 9:1; 8:1, 7:1, 6:1,5:1, 4:1, 3:1, 2:1, or 1:1, or in a ratio of (white bloodcells:apoptotic cells) 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.In one example, the ratio of white blood cells to apoptotic cells is1:5.

The co-culture of the cells might be for several hours to several days.In some embodiments, said apoptotic cells are cultured for 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52 hours. A person skilled in the art can evaluate theoptimal time for co-culture by measuring the presence ofanti-inflammatory compounds, the viable amount of white blood cells andthe amount of apoptotic cells which have not been eliminated so far.elimination of apoptotic cells by phagocytes is observable with lightmicroscopy due to the disappearance of apoptotic cells.

In some embodiments, the culture of apoptotic cells, such as theco-culture with culture with white blood cells (e.g. phagocytes such asmacrophages, monocytes, or phagocytes, or the B, T or NK cells), takesplace in culture medium and/or in a physiological solution compatiblewith administration e.g. injection to a subject.

A skilled artisan would appreciate that a “physiological solution” mayencompass a solution which does not lead to the death of white bloodcells within the culture time. In some embodiments, the physiologicalsolution does not lead to death over 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 hours. Inother embodiment, 48 hours, or 30 hours.

In some embodiments, the white blood cells (e.g. phagocytes such asmacrophages, monocytes, or phagocytes, or the B, T or NK cells) and theapoptotic cells are incubated in the physiological solution for at least30 min. This time of culture allows phagocytosis initiation andsecretion of cytokines and other beneficial substances.

In an embodiment, such a physiological solution does not inhibitapoptotic leukocyte elimination by leukocyte-derived macrophages.

At the end of the culture or the co-culture step, the supernatant isoptionally separated from the cultured apoptotic cells or theco-cultured cells. Techniques to separate the supernatant from the cellsare known in the art. For example, the supernatant can be collectedand/or filtered and/or centrifuged to eliminate cells and debris. Forexample, said supernatant may be centrifuged at 3000 rpm for 15 minutesat room temperature to separate it from the cells.

The supernatant may be “inactivated” prior to use, for example byirradiation. Therefore, the method for preparing the apoptotic cellsupernatant may comprise an optional additional irradiation step f).Said “irradiation” step can be considered as a disinfection method thatuses X-ray irradiation (25-45 Gy) at sufficiently rate to killmicroorganisms, as routinely performed to inactivate blood products.

Irradiation of the supernatant is considered safe in the art.Irradiation procedures are currently performed on a routine basis todonated blood to prevent reactions to WBC.

In an embodiment, the apoptotic cell supernatant is formulated into apharmaceutical composition suitable for administration to a subject, asdescribed in detail herein.

In some embodiments, the final product is stored at +4° C. In anotherembodiment, the final product is for use in the next 48 hours.

In some embodiments, the apoptotic cell supernatant, such as anapoptotic cell-phagocyte supernatant, or pharmaceutical compositioncomprising the supernatant, may be lyophilized, for example for storageat −80° C.

In one specific embodiment, as described in Example 1 of WO 2014/106666,an apoptotic cell-phagocyte supernatant may be made using thymic cellsas apoptotic cells. After isolation, thymic cells are irradiated (e.g.with a 35 X-Gray irradiation) and cultured in complete DMEM culturemedium for, for example, 6 hours to allow apoptosis to occur. Inparallel, macrophages are isolated from the peritoneum cavity, washedand cultured in complete RPMI (10% FBS, Peni-Strepto, EAA, Hepes, NaPand 2-MercaptoEthanol). Macrophages and apoptotic cells are then washedand co-cultured for another 48 hour period in phenol-free X-vivo mediumat a 1/5 macrophage/apoptotic cell ratio. Then, supernatant iscollected, centrifuged to eliminate debris and may be frozen orlyophilized for conservation. Macrophage enrichment may be confirmedusing positive staining for F4/80 by FACS. Apoptosis may be confirmed byFACS using positive staining for Annexin-V and 7AAD exclusion.

In an embodiment, the apoptotic cell supernatant is enriched in TGF-βlevels both in active and latent forms of TGF-β, compared tosupernatants obtained from either macrophages or apoptotic cellscultured separately. In an embodiment, IL-1β levels are also increasedcompared to macrophages cultured alone and dramatically increasedcompared to apoptotic cells cultured alone. In another embodiment,inflammatory cytokines such as IL-6 are not detectable and IL-1β and TNFare undetectable or at very low levels.

In an embodiment, the apoptotic cell supernatant, when compared tosupernatants from macrophages cultured alone or from apoptotic cellscultured alone, has increased levels of IL-ira, TIMP-1, CXCL1/KC andCCL2/JE/MCP1, which might be implicated in a tolerogenic role of thesupernatant to control inflammation, in addition to TGF-β and IL-10.

In another specific embodiment, as described in Example 3 of WO2014/106666, human apoptotic cell-phagocyte supernatant may be made fromthe co-culture of macrophages derived from peripheral blood mononuclearcells (PBMC) cultured with apoptotic PBMC. Thus, PBMC are isolated fromcytapheresis bag from a healthy volunteer through, for example, Ficollgradient centrifugation. Then PBMC are plated for 90 min in completeRPMI culture medium (10% FBS, 1% Penicillin/Streptomycin). Then,non-adherenT-cells are removed and rendered apoptotic using, forexample, a 35 Gy dose of X-ray irradiation and cultured in complete RPMImilieu for 4 days (including cell wash after the first 48 hrs ofculture), in order to allow apoptosis to occur. In parallel, adherentT-cells are cultured in complete RPMI milieu supplemented with 50 μg/mLof recombinant human M-CSF for 4 days including cell wash after thefirst 48 hrs. At the end of the 4-day culture period, monocyte-derivedmacrophages and apoptotic cells are washed and cultured together inX-vivo medium for again 48 hours at a one macrophage to 5 apoptotic cellratio. Then supernatant from the latter culture is collected,centrifuged to eliminate cells and debris, and may be frozen orlyophilized for conservation and subsequent use.

In an embodiment, as described in WO 2014/106666, human apoptoticcell-phagocyte supernatant may be obtained in 6 days from peripheralblood mononuclear cells (PBMC). Four days to obtain PBMC-derivedmacrophages using M-CSF addition in the culture, and 2 more days for theco-culture of PBMC-derived macrophages with apoptotic cells,corresponding to the non-adherent PBMC isolated at day 0.

In an embodiment, as described in WO 2014/106666, a standardized humanapoptotic cell-phagocyte supernatant may be obtained independently ofthe donor or the source of PBMC (cytapheresis or buffy coat). Theplastic-adherence step is sufficient to obtain a significant startingpopulation of enriched monocytes (20 to 93% of CD14+ cells afteradherence on plastic culture dish). In addition, such adherent T-cellsdemonstrate a very low presence of B and T-cells (1.0% of CD19+ B cellsand 12.8% of CD3+ T-cells). After 4 days of culture of adherent T-cellsin the presence of M-CSF, the proportion of monocytesderived-macrophages is significantly increased from 0.1% to 77.7% ofCD14+CD206+HLA-DR+ macrophages. At that time, monocyte-derivedmacrophages may be co-cultured with apoptotic non-adherent PBMC (47.6%apoptotic as shown by annexin V staining and 7AAD exclusion) to producethe apoptotic cell-phagocyte supernatant during 48 hours.

In an embodiment, the collected apoptotic cell-phagocyte supernatant,contains significantly more latent TGF than in the culture supernatantof monocyte-derived macrophages alone or monocyte-derived macrophagestreated in inflammatory conditions (+LPS), and only contains trace orlow level of inflammatory cytokines such as IL-1β or TNF.

In some embodiments, the composition comprising the apoptotic cellsupernatant further comprises an anti-coagulant. In some embodiments,the anti-coagulant is selected from the group consisting of: heparin,acid citrate dextrose (ACD) Formula A and a combination thereof.

In another embodiment, an anti-coagulant is added during the process ofmanufacturing apoptotic cells. In another embodiment, the anti-coagulantadded is selected from the group comprising ACD and heparin, or anycombination thereof. In another embodiment, ACD is at a concentration of1%. In another embodiment, ACD is at a concentration of 2%. In anotherembodiment, ACD is at a concentration of 3%. In another embodiment, ACDis at a concentration of 4%. In another embodiment, ACD is at aconcentration of 5%. In another embodiment, ACD is at a concentration of6%. In another embodiment, ACD is at a concentration of 7%. In anotherembodiment, ACD is at a concentration of 8%. In another embodiment, ACDis at a concentration of 9%. In another embodiment, ACD is at aconcentration of 10%. In another embodiment, ACD is at a concentrationof between about 1-10%. In another embodiment, ACD is at a concentrationof between about 2-8%. In another embodiment, ACD is at a concentrationof between about 3-7%. In another embodiment, ACD is at a concentrationof between about 1-5%. In another embodiment, ACD is at a concentrationof between about 5-10%. In another embodiment, heparin is at a finalconcentration of 0.5 U/ml. In another embodiment, heparin is at a finalconcentration of about 0.1 U/ml-1.0 U/ml. In another embodiment, heparinis at a final concentration of about 0.2 U/ml-0.9 U/ml. In anotherembodiment, heparin is at a final concentration of about 0.3 U/ml-0.7U/ml. In another embodiment, heparin is at a final concentration ofabout 0.1 U/ml-0.5 U/ml. In another embodiment, heparin is at a finalconcentration of about 0.5 U/ml-1.0 U/ml. In another embodiment, heparinis at a final concentration of about 0.01 U/ml-1.0 U/ml. In anotherembodiment, heparin is at a final concentration of 0.1 U/ml. In anotherembodiment, heparin is at a final concentration of 0.2 U/ml. In anotherembodiment, heparin is at a final concentration of 0.3 U/ml. In anotherembodiment, heparin is at a final concentration of 0.4 U/ml. In anotherembodiment, heparin is at a final concentration of 0.5 U/ml. In anotherembodiment, heparin is at a final concentration of 0.6 U/ml. In anotherembodiment, heparin is at a final concentration of 0.7 U/ml. In anotherembodiment, heparin is at a final concentration of 0.8 U/ml. In anotherembodiment, heparin is at a final concentration of 0.9 U/ml. In anotherembodiment, heparin is at a final concentration of 1.0 U/ml. In anotherembodiment, ACD is at a concentration of 5% and heparin is at a finalconcentration of 0.5 U/ml.

In some embodiments, the composition comprising the apoptotic cellsupernatant further comprises methylprednisolone. At some embodiments,the concentration of methylprednisolone does not exceed 30 μg/ml.

In some embodiments, the composition may be used at a total dose oraliquot of apoptotic cell supernatant derived from the co-culture ofabout 14×10⁹ of CD45+ cells obtained by cytapheresis equivalent to about200 million of cells per kilogram of body weight (for a 70 kg subject).In an embodiment, such a total dose is administered as unit doses ofsupernatant derived from about 100 million cells per kilogram bodyweight, and/or is administered as unit doses at weekly intervals, Inanother embodiment both of which. Suitable total doses according to thisembodiment include total doses of supernatant derived from about 10million to about 4 billion cells per kilogram body weight. In anotherembodiment, the supernatant is derived from about 40 million to about 1billion cells per kilogram body weight. In yet another embodiment thesupernatant is derived from about 80 million to about 500 million cellsper kilogram body weight. In still another embodiment, the supernatantis derived from about 160 million to about 250 million cells perkilogram body weight. Suitable unit doses according to this embodimentinclude unit doses of supernatant derived from about 4 million to about400 million cells per kilogram body weight. In another embodiment, thesupernatant is derived from about 8 million to about 200 million cellsper kilogram body weight. In another embodiment, the supernatant isderived from about 16 million to about 100 million cells per kilogrambody weight. In yet another embodiment, the supernatant is derived fromabout 32 million to about 50 million cells per kilogram body weight.

In another embodiment, a dose of apoptotic cell supernatant derived fromthe co-culture of about 10×10⁶ apoptotic cells is administered. Inanother embodiment, a dose derived from 10×10⁷ apoptotic cells isadministered. In another embodiment, a dose derived from 10×10⁸apoptotic cells is administered. In another embodiment, a dose derivedfrom 10×10⁹ apoptotic cells is administered. In another embodiment, adose derived from 10×10¹⁰ apoptotic cells is administered. In anotherembodiment, a dose derived from 10×10¹¹ apoptotic cells is administered.In another embodiment, a dose derived from 10×10¹² apoptotic cells isadministered. In another embodiment, a dose derived from 10×10⁵apoptotic cells is administered. In another embodiment, a dose derivedfrom 10×10⁴ apoptotic cells is administered. In another embodiment, adose derived from 10×10³ apoptotic cells is administered. In anotherembodiment, a dose derived from 10×10² apoptotic cells is administered.

In some embodiments, a dose of apoptotic cell supernatant derived from35×10⁶ apoptotic cells is administered. In another embodiment, a dosederived from 210×10⁶ apoptotic cells is administered. In anotherembodiment, a dose derived from 70×10⁶ apoptotic cells is administered.In another embodiment, a dose derived from 140×10⁶ apoptotic cells isadministered. In another embodiment, a dose derived from 35-210×10⁶apoptotic cells is administered.

In some embodiments, the apoptotic cell supernatant, or compositioncomprising said apoptotic cell supernatant, may be administered by anymethod known in the art including, but not limited to, intravenous,subcutaneous, intranodal, intratumoral, intrathecal, intrapleural,intraperitoneal and directly to the thymus, as discussed in detailherein.

Surprisingly, the apoptotic cell supernatants, such as apoptoticcell-phagocyte supernatants, reduces production of cytokines associatedwith the cytokine storm such as IL-6. Another cytokine, IL-2, is notinvolved in cytokine release syndrome although is secreted by DCs andmacrophages in small quantities. It is, however, required for thesurvival and proliferation of CAR-T-cells and is mostly produced bythese T-cells. Unexpectedly, the apoptotic cell supernatants, such asapoptotic cell-phagocyte supernatants, do not reduce IL-2 levelssufficiently to negatively affect the survival of CAR T-cells.

In some embodiments, the apoptotic cell supernatants, such as apoptoticcell-phagocyte supernatants, affect cytokine expression levels inmacrophages and DCs, but do not affect cytokine expression levels in theT-cells themselves. It was therefore unexpected that apoptotic cellsupernatants would be useful in enhancing CAR T-cell therapy ordendritic cell therapy.

In another embodiment, the apoptotic cell supernatants trigger death ofT-cells, but not via changes in cytokine expression levels.

In another embodiment, apoptotic cell supernatants, such as apoptoticcell-phagocyte supernatants antagonize the priming of macrophages anddendritic cells to secrete cytokines that would otherwise amplify thecytokine storm. In another embodiment, apoptotic cell supernatantsincrease Tregs which suppress the inflammatory response and/or preventexcess release of cytokines.

In some embodiments, administration of apoptotic cell supernatants, suchas apoptotic cell-phagocyte supernatants, inhibits one or morepro-inflammatory cytokines. In some embodiments, the pro-inflammatorycytokine comprises IL-1beta, IL-6, TNF-alpha, or IFN-gamma, or anycombination thereof. In another embodiment, administration of apoptoticcell supernatants promotes the secretion of one or moreanti-inflammatory cytokines. In some embodiments, the anti-inflammatorycytokine comprises TGF-beta, IL10, or PGE2, or any combination thereof.

In another embodiment, administration of apoptotic cell supernatants,such as apoptotic cell-phagocyte supernatants, inhibits dendritic cellmaturation following exposure to TLR ligands. In another embodiment,administration of apoptotic cell supernatants creates potentiallytolerogenic dendritic cells, which in some embodiments, are capable ofmigration, and in some embodiments, the migration is due to CCR7. Inanother embodiment, administration of apoptotic cell supernatantselicits various signaling events which in one embodiment is TAM receptorsignaling (Tyro3, Axl and Mer) which in some embodiments, inhibitsinflammation in antigen-presenting cells. In some embodiments, Tyro-3,Axl, and Mer constitute the TAM family of receptor tyrosine kinases(RTKs) characterized by a conserved sequence within the kinase domainand adhesion molecule-like extracellular domains. In another embodiment,administration of apoptotic cell supernatants activates signalingthrough MerTK. In another embodiment, administration of apoptotic cellsupernatants activates the phosphatidylinositol 3-kinase (PI3K)/AKTpathway, which in some embodiments, negatively regulates NF-κB. Inanother embodiment, administration of apoptotic cell supernatantsnegatively regulates the inflammasome which in one embodiment leads toinhibition of pro-inflammatory cytokine secretion, DC maturation, or acombination thereof. In another embodiment, administration of apoptoticcell supernatants upregulates expression of anti-inflammatory genes suchas Nr4a, Thbs1, or a combination thereof. In another embodiment,administration of apoptotic cell supernatants induces a high level ofAMP which in some embodiments, is accumulated in a Pannexin1-dependentmanner. In another embodiment, administration of apoptotic cellsupernatants suppresses inflammation.

Compositions

As used herein, the terms “composition” and pharmaceutical composition”may in some embodiments, be used interchangeably having all the samequalities and meanings. In some embodiments, disclosed herein is apharmaceutical composition for the treatment of a condition or diseaseas described herein.

In another embodiment, pharmaceutical compositions disclosed here arefor maintaining or increasing the proliferation rate of a geneticallymodified immune cells. In a further embodiment, methods for maintainingor increasing the proliferation rate of genetically modified immunecells further comprise reducing or inhibiting the incidence of cytokinerelease syndrome (CRS) or cytokine storm. In another embodiment,disclosed herein are pharmaceutical compositions for increasing theefficacy of a genetically modified immune cell therapy. In anotherembodiment, compositions used in the methods for increasing the efficacyof an immune cell therapy further comprise reducing or inhibiting theincidence of CRS or a cytokine storm. In another embodiment, disclosedherein are compositions for methods treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating a cancer of atumor in a subject. In another embodiment, compositions used in themethods for treating, preventing, reducing the incidence of,ameliorating, or alleviating a cancer or a tumor in a subject, furthercomprise reducing or inhibiting the incidence of CRS or a cytokinestorm.

In another embodiment, a pharmaceutical composition comprises agenetically modified immune cell or a genetically modified receptorthereof. In another embodiment, a genetically modified immune cellcomprises a T-cell. In another embodiment, a genetically modified immunecell comprises a chimeric antigen receptor CAR T-cell. In anotherembodiment, a genetically modified immune cell comprises a chimericantigen receptor TCR T-cell. In another embodiment, a geneticallymodified immune cell comprises a cytotoxic T lymphocyte. In anotherembodiment, a genetically modified immune cell comprises a dendriticcell. In another embodiment, a genetically modified immune cellcomprises a natural killer cell. In another embodiment, a geneticallymodified receptor comprises a genetically modified T-cell receptor.

In another embodiment, a pharmaceutical composition comprises an earlyapoptotic cell population. In another embodiment, a pharmaceuticalcomposition comprises an apoptotic supernatant.

In still another embodiment, a pharmaceutical composition for thetreatment of a condition or a disease as described herein comprises aneffective amount of a genetically modified immune cell or a geneticallymodified receptor thereof, as described herein in a pharmaceuticallyacceptable excipient. In another embodiment, a pharmaceuticalcomposition for the treatment of a condition or a disease as describedherein comprises an effective amount of a CAR T-cell as described hereinin, and a pharmaceutically acceptable excipient. In another embodiment,a pharmaceutical composition for the treatment of a condition or adisease as described herein comprises an effective amount of a TCRT-cell as described herein in, and a pharmaceutically acceptableexcipient. In another embodiment, a pharmaceutical composition for thetreatment of a condition or a disease as described herein comprises aneffective amount of a cytotoxic T-cell, as described herein, and apharmaceutically acceptable excipient. In another embodiment, apharmaceutical composition for the treatment of a condition or a diseaseas described herein comprises an effective amount of a geneticallymodified dendritic cell, as described herein, and a pharmaceuticallyacceptable excipient. In another embodiment, a pharmaceuticalcomposition for the treatment of a condition or a disease as describedherein comprises an effective amount of a genetically modified naturalkiller cell, as described herein, and a pharmaceutically acceptableexcipient. In another embodiment, a pharmaceutical composition for thetreatment of a condition or a disease as described herein comprises aneffective amount of a genetically modified T-cell receptor, as describedherein, and a pharmaceutically acceptable excipient. In still anotherembodiment, a pharmaceutical composition for the treatment of acondition or a disease as described herein comprises an effective amountof an early apoptotic cell population, as described herein in apharmaceutically acceptable excipient. In still another embodiment, apharmaceutical composition for the treatment of a condition or a diseaseas described herein comprises an effective amount of an apoptoticsupernatant, as described herein in a pharmaceutically acceptableexcipient.

In another embodiment, the condition or disease as described herein is atumor or cancer. In another embodiment, disclosed herein is acomposition comprising the genetically modified immune cell or receptorthereof, for example a CAR T-cell, that binds to a protein or peptide ofinterest as described herein. In another embodiment, disclosed herein isa composition comprising the genetically modified immune cell orreceptor thereof, for example a TCR T-cell, that recognizes and binds aprotein or peptide of interest as described herein. In anotherembodiment, the protein or peptide of interest comprises a tumor antigenor a fragment thereof.

In another embodiment, a composition disclosed herein and used inmethods disclosed herein comprises apoptotic cells or an apoptotic cellsupernatant, and a pharmaceutically acceptable excipient. In someembodiments, a composition comprising apoptotic cells or an apoptoticcell supernatant is used in methods disclosed herein for example fortreating, preventing, inhibiting the growth of, delaying diseaseprogression, reducing the tumor load, or reducing the incidence of acancer or a tumor in a subject, or any combination thereof.

In yet another embodiment, a composition comprising an effective amountof a genetically modified immune cell or a genetically modified receptorthereof may be the same composition as comprises an apoptotic cellpopulation or an apoptotic cell supernatant. In another embodiment, acomposition comprising an effective amount of a CAR T-cell, or a TCRT-cell, or a cytotoxic T-cell, or a genetically modified dendritic cell,or a genetically modified natural killer cell may be the samecomposition as comprises an apoptotic cell population or an apoptoticcell supernatant. In yet another embodiment, a composition comprising aneffective amount of genetically modified T-cell receptor may be the samecomposition as comprises an apoptotic cell population or an apoptoticcell supernatant. In still another embodiment, a composition comprisingan effective amount of a genetically modified immune cell selected fromthe group comprising a CAR T-cell, a TCR T-cell, a cytotoxic T-cell, anatural killer cell, or a dendritic cell, is not the same composition ascomprises an apoptotic cell population or an apoptotic cell supernatant.In another embodiment, a composition comprises a chimeric antigenreceptor-expressing T-cell (CAR T-cell) and either apoptotic cells or anapoptotic cell supernatant, and a pharmaceutically acceptable excipient.In another embodiment, a composition comprises a genetically modifiedT-cell receptor expressing T-cell (TCR T-cell) and either apoptoticcells or an apoptotic cell supernatant, and a pharmaceuticallyacceptable excipient. In another embodiment, a composition comprising aneffective amount of a genetically modified T-cell receptor is not thesame composition as comprises an apoptotic cell population or anapoptotic cell supernatant.

In another embodiment, apoptotic cells comprised in a compositioncomprise apoptotic cells in an early apoptotic state. In anotherembodiment, apoptotic cells comprised in a composition are pooled thirdparty donor cells. In another embodiment, an apoptotic cell supernatantcomprised in a composition disclosed herein is collected from earlyapoptotic cells. In another embodiment, an apoptotic cell supernatantcomprised in a composition disclosed herein, is collected pooled thirdparty donor cells.

In one embodiment, a composition comprising a genetically modifiedimmune cells, for example a CAR T-cell, further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a CART-cell, and apoptotic cells further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a CART-cell, and an apoptotic cell supernatant, further comprises anadditional pharmaceutical composition for preventing, suppressing, ormodulating cytokine release in a patient with cytokine release syndromeor experiencing a cytokine storm.

In one embodiment, a composition comprising a genetically modifiedimmune cells, for example a TCR T-cell, further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a TCRT-cell, and apoptotic cells further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a TCRT-cell, and an apoptotic cell supernatant, further comprises anadditional pharmaceutical composition for preventing, suppressing, ormodulating cytokine release in a patient with cytokine release syndromeor experiencing a cytokine storm.

In one embodiment, a composition comprising a genetically modifiedimmune cells, for example a dendritic cell, further comprises anadditional pharmaceutical composition for preventing, suppressing, ormodulating cytokine release in a patient with cytokine release syndromeor experiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a dendritic,and apoptotic cells further comprises an additional pharmaceuticalcomposition for preventing, suppressing, or modulating cytokine releasein a patient with cytokine release syndrome or experiencing a cytokinestorm. In another embodiment, a composition comprising a geneticallymodified immune cells, for example a dendritic, and an apoptotic cellsupernatant, further comprises an additional pharmaceutical compositionfor preventing, suppressing, or modulating cytokine release in a patientwith cytokine release syndrome or experiencing a cytokine storm.

In one embodiment, a composition comprising a genetically modifiedimmune cells, for example a NK cell, further comprises an additionalpharmaceutical composition for preventing, suppressing, or modulatingcytokine release in a patient with cytokine release syndrome orexperiencing a cytokine storm. In another embodiment, a compositioncomprising a genetically modified immune cells, for example a NK cell,and apoptotic cells further comprises an additional pharmaceuticalcomposition for preventing, suppressing, or modulating cytokine releasein a patient with cytokine release syndrome or experiencing a cytokinestorm. In another embodiment, a composition comprising a geneticallymodified immune cells, for example a NK cell, and an apoptotic cellsupernatant, further comprises an additional pharmaceutical compositionfor preventing, suppressing, or modulating cytokine release in a patientwith cytokine release syndrome or experiencing a cytokine storm.

In one embodiment, the additional pharmaceutical composition comprises aCTLA-4 blocking agent, which in one embodiment is Ipilimumab. In anotherembodiment, the additional pharmaceutical composition comprises aalpha-1 anti-trypsin, as disclosed herein, or a fragment thereof, or ananalogue thereof. In another embodiment, the additional pharmaceuticalcomposition comprises a tellurium-based compound, a disclosed herein. Inanother embodiment, the additional pharmaceutical composition comprisesan immune modulating agent, as disclosed herein. In another embodiment,the additional pharmaceutical composition comprises a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment thereof or analogue thereof,a tellurium-based compound, or an immune modulating compound, or anycombination thereof.

In one embodiment, the composition comprising the genetically modifiedimmune cell, for example a CAR T-cell and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, or anapoptotic cell supernatant, a tellurium-based compound, or an immunemodulating agent comprises a single composition. In another embodiment,the composition comprising the genetically modified immune cell, forexample CAR T-cells and the pharmaceutical composition comprising anyone of a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragmentthereof or analogue thereof, apoptotic cells, or an apoptotic cellsupernatant, a tellurium-based compound, or an immune modulating agent,or any combination thereof, comprises multiple compositions, whereineach of the genetically modified immune cell, which in one embodiment isCAR T-cells, the CTLA-4 blocking agent, the alpha-1 anti-trypsin orfragment thereof or analogue thereof, the apoptotic cells, the apoptoticcell supernatant, the tellurium-based compound, or the immune modulatingagent, or any combination thereof, are comprised in a separatecomposition. In yet another embodiment, the composition comprising thegenetically modified immune cell, which in one embodiment is CAR T-cellsand the pharmaceutical composition comprising any one of a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, apoptotic cells, an apoptotic cell supernatant, atellurium-based compound, or an immune modulating agent, or anycombination thereof, comprises multiple compositions, wherein thegenetically modified immune cells, which in one embodiment are CART-cells, the CTLA-4 blocking agent, or the alpha-1 anti-trypsin orfragment thereof or analogue thereof, the tellurium-based compound, orthe immune modulating agent, or any combination thereof, or anycombination thereof are present in the genetically modified immune cell,for example a CAR T-cell, composition, and the apoptotic cells, or theapoptotic cell supernatant, are comprised in a separate composition.

In one embodiment, the composition comprising the genetically modifiedimmune cell, for example a TCR T-cell and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, or anapoptotic cell supernatant, a tellurium-based compound, or an immunemodulating agent comprises a single composition. In another embodiment,the composition comprising the genetically modified immune cell, forexample TCR T-cells and the pharmaceutical composition comprising anyone of a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragmentthereof or analogue thereof, apoptotic cells, or an apoptotic cellsupernatant, a tellurium-based compound, or an immune modulating agent,or any combination thereof, comprises multiple compositions, whereineach of the genetically modified immune cell, which in one embodiment isTCR T-cells, the CTLA-4 blocking agent, the alpha-1 anti-trypsin orfragment thereof or analogue thereof, the apoptotic cells, the apoptoticcell supernatant, the tellurium-based compound, or the immune modulatingagent, or any combination thereof, are comprised in a separatecomposition. In yet another embodiment, the composition comprising thegenetically modified immune cell, which in one embodiment is TCR T-cellsand the pharmaceutical composition comprising any one of a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, apoptotic cells, an apoptotic cell supernatant, atellurium-based compound, or an immune modulating agent, or anycombination thereof, comprises multiple compositions, wherein thegenetically modified immune cells, which in one embodiment are TCRT-cells, the CTLA-4 blocking agent, or the alpha-1 anti-trypsin orfragment thereof or analogue thereof, the tellurium-based compound, orthe immune modulating agent, or any combination thereof, or anycombination thereof are present in the genetically modified immune cell,for example a TCR T-cell, composition, and the apoptotic cells, or theapoptotic cell supernatant, are comprised in a separate composition.

In one embodiment, the composition comprising the genetically modifiedimmune cell, for example a dendritic cell and the pharmaceuticalcomposition comprising any one of a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment thereof or analogue thereof, apoptotic cells,or an apoptotic cell supernatant, a tellurium-based compound, or animmune modulating agent comprises a single composition. In anotherembodiment, the composition comprising the genetically modified immunecell, for example dendritic cells and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, or anapoptotic cell supernatant, a tellurium-based compound, or an immunemodulating agent, or any combination thereof, comprises multiplecompositions, wherein each of the genetically modified immune cell,which in one embodiment is dendritic cells, the CTLA-4 blocking agent,the alpha-1 anti-trypsin or fragment thereof or analogue thereof, theapoptotic cells, the apoptotic cell supernatant, the tellurium-basedcompound, or the immune modulating agent, or any combination thereof,are comprised in a separate composition. In yet another embodiment, thecomposition comprising the genetically modified immune cell, which inone embodiment is dendritic cells and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, an apoptoticcell supernatant, a tellurium-based compound, or an immune modulatingagent, or any combination thereof, comprises multiple compositions,wherein the genetically modified immune cells, which in one embodimentare dendritic cells, the CTLA-4 blocking agent, or the alpha-1anti-trypsin or fragment thereof or analogue thereof, thetellurium-based compound, or the immune modulating agent, or anycombination thereof, or any combination thereof are present in thegenetically modified immune cell, for example a dendritic cell,composition, and the apoptotic cells, or the apoptotic cell supernatant,are comprised in a separate composition.

In one embodiment, the composition comprising the genetically modifiedimmune cell, for example a NK cell and the pharmaceutical compositioncomprising any one of a CTLA-4 blocking agent, an alpha-1 anti-trypsinor fragment thereof or analogue thereof, apoptotic cells, or anapoptotic cell supernatant, a tellurium-based compound, or an immunemodulating agent comprises a single composition. In another embodiment,the composition comprising the genetically modified immune cell, forexample NK cells and the pharmaceutical composition comprising any oneof a CTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment thereofor analogue thereof, apoptotic cells, or an apoptotic cell supernatant,a tellurium-based compound, or an immune modulating agent, or anycombination thereof, comprises multiple compositions, wherein each ofthe genetically modified immune cell, which in one embodiment is NKcells, the CTLA-4 blocking agent, the alpha-1 anti-trypsin or fragmentthereof or analogue thereof, the apoptotic cells, the apoptotic cellsupernatant, the tellurium-based compound, or the immune modulatingagent, or any combination thereof, are comprised in a separatecomposition. In yet another embodiment, the composition comprising thegenetically modified immune cell, which in one embodiment is NK cellsand the pharmaceutical composition comprising any one of a CTLA-4blocking agent, an alpha-1 anti-trypsin or fragment thereof or analoguethereof, apoptotic cells, an apoptotic cell supernatant, atellurium-based compound, or an immune modulating agent, or anycombination thereof, comprises multiple compositions, wherein thegenetically modified immune cells, which in one embodiment are NK cells,the CTLA-4 blocking agent, or the alpha-1 anti-trypsin or fragmentthereof or analogue thereof, the tellurium-based compound, or the immunemodulating agent, or any combination thereof, or any combination thereofare present in the genetically modified immune cell, for example a NKcell, composition, and the apoptotic cells, or the apoptotic cellsupernatant, are comprised in a separate composition.

In some embodiments, a composition comprises apoptotic cells and anadditional agent. In some embodiments, a composition comprises apoptoticcells and an antibody or a functional fragment thereof. In someembodiments, a composition comprises apoptotic cells and a RtX antibodyor a functional fragment thereof. In some embodiments, apoptotic cellsand an antibody or a functional fragment thereof may be comprised inseparate compositions. In some embodiments, apoptotic cells and anantibody or a functional fragment thereof may be comprised in the samecomposition.

A skilled artisan would appreciate that a “pharmaceutical composition”may encompass a preparation of one or more of the active ingredientsdescribed herein with other chemical components such as physiologicallysuitable carriers and excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

In some embodiments, disclosed herein is a pharmaceutical compositionfor treating, preventing, inhibiting the growth of, or reducing theincidence of a cancer or a tumor. In some embodiments, disclosed hereinis a pharmaceutical composition for increasing the survival of a subjectsuffering from a cancer or a tumor. In some embodiments, disclosedherein is a pharmaceutical composition for reducing the size or reducingthe growth rate of a tumor or a cancer. In some embodiments, disclosedherein is a pharmaceutical comprising for reducing tumor load in asubject suffering from a cancer or a tumor. In some embodiments,disclosed herein is a pharmaceutical comprising for delaying diseaseprogression in a subject suffering from a cancer or a tumor. In someembodiments, disclosed herein is a pharmaceutical comprising forreducing the incidence of cancer or a tumor in a subject suffering froma cancer or a tumor. In some embodiments, disclosed herein is apharmaceutical comprising for reducing the size and or growth rate of acancer or tumor in a subject suffering from a cancer or a tumor.

In some embodiments, a pharmaceutical composition comprises an earlyapoptotic cell population as described herein. In some embodiments, apharmaceutical composition comprises an early apoptotic cell populationas described herein, and a pharmaceutically acceptable excipient.

A skilled artisan would appreciate that the phrases “physiologicallyacceptable carrier”, “pharmaceutically acceptable carrier”,“physiologically acceptable excipient”, and “pharmaceutically acceptableexcipient”, may be used interchangeably may encompass a carrier,excipient, or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered active ingredient.

A skilled artisan would appreciate that an “excipient” may encompass aninert substance added to a pharmaceutical composition to furtherfacilitate administration of an active ingredient. In some embodiments,excipients include calcium carbonate, calcium phosphate, various sugarsand types of starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

In some embodiments, compositions are administered at the same time. Inan alternative embodiment, compositions are administered at differenttimes. In another embodiment, compositions comprising apoptotic cellsare administered prior to infusion or genetically modified immune cellsor receptors thereof. In another embodiment, compositions comprisingapoptotic cells are administered prior to CAR-T-cell infusion. Inanother embodiment, compositions comprising apoptotic cells areadministered prior to cytotoxic T-cell infusion. In another embodiment,compositions comprising apoptotic cells are administered prior tonatural killer cell infusion. In another embodiment, compositionscomprising apoptotic cells are administered prior to dendritic infusion.In another embodiment, compositions comprising apoptotic cells areadministered prior to infusion of a genetically modified T-cellreceptor.

In another embodiment, compositions comprising apoptotic cellsupernatants are administered prior to infusion or genetically modifiedimmune cells or receptors thereof. In another embodiment, compositionscomprising apoptotic cell supernatants are administered prior toCAR-T-cell infusion. In another embodiment, compositions comprisingapoptotic cell supernatants are administered prior to cytotoxic T-cellinfusion. In another embodiment, compositions comprising apoptotic cellsupernatants are administered prior to natural killer cell infusion. Inanother embodiment, compositions comprising apoptotic cell supernatantsare administered prior to dendritic infusion. In another embodiment,compositions comprising apoptotic cell supernatants are administeredprior to infusion of a genetically modified T-cell receptor.

In another embodiment, compositions comprising apoptotic cellsupernatants are administered prior to infusion of genetically modifiedimmune cells or receptors thereof. In another embodiment, compositionscomprising apoptotic cells are administered about 24 hours prior togenetically modified immune cell or receptor thereof infusion. Inanother embodiment, compositions comprising apoptotic cells areadministered about 24 hours prior to CAR T-cell, or cytotoxic T-cells,or natural killer cells, or dendritic cell or genetically modifiedT-cell receptor infusion. In another embodiment, compositions comprisingapoptotic cell supernatants are administered about 24 hours prior to CART-cell or cytotoxic T-cells, or natural killer cells, or dendritic cellor genetically modified T-cell receptor infusion.

In some embodiments, compositions are administered at the same time. Inan alternative embodiment, compositions are administered at differenttimes. In another embodiment, compositions comprising apoptotic cellsare administered prior to administration of an antibody or fragmentthereof, or a composition comprising an antibody or fragment thereof.

In another embodiment, compositions comprising apoptotic cells areadministered about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12hours, 14 hours, 16 hours, 18 hours 20 hours, 22 hours, 24 hours, 36hours, 48 hours, 60 hours, or 72 hours prior to an antibody or fragmentthereof, or composition comprising the antibody or fragment thereof. Inanother embodiment, compositions comprising apoptotic cell supernatantsare administered about 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12hours, 14 hours, 16 hours, 18 hours 20 hours, 22 hours, 24 hours, 36hours, 48 hours, 60 hours, or 72 hours prior to an antibody or fragmentthereof, or composition comprising the antibody or fragment thereof.

In another embodiment, compositions comprising apoptotic cells areadministered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days 14 days, or 15days prior to an antibody or fragment thereof, or composition comprisingthe antibody or functional fragment thereof. In another embodiment,compositions comprising apoptotic cell supernatants are administeredabout 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeksprior to an antibody or functional fragment thereof, or compositioncomprising the antibody or functional fragment thereof.

In another embodiment, compositions comprising apoptotic cells areadministered after infusion of an antibody or fragment thereof, orcomposition comprising the antibody or fragment thereof. In anotherembodiment, composition comprising apoptotic cells are administeredafter an antibody or fragment thereof, or composition comprising theantibody or fragment thereof. In another embodiment, compositionscomprising apoptotic cell supernatants are administered afteradministration of an antibody or fragment thereof, or compositioncomprising the antibody or fragment thereof. In another embodiment,compositions comprising apoptotic cell supernatants are administeredafter administration of an antibody or fragment thereof, or compositioncomprising the antibody or fragment thereof. In another embodiment,compositions comprising apoptotic cells are administered about 24 hoursafter an antibody or fragment thereof, or composition comprising theantibody or fragment thereof. In another embodiment, compositionscomprising apoptotic cells are administered after administration of anantibody or fragment thereof, or composition comprising the antibody orfragment thereof. In another embodiment, compositions comprisingapoptotic cells are administered about 2 hours, 4 hours, 6 hours, 8hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours 20 hours, 22hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours afteradministration of an antibody or fragment thereof, or compositioncomprising the antibody or fragment thereof. In another embodiment,compositions comprising apoptotic cell supernatants are administeredabout 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours,16 hours, 18 hours 20 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60hours, or 72 hours after administration of an antibody or fragmentthereof, or composition comprising the antibody or fragment thereof.

In another embodiment, compositions comprising apoptotic cells areadministered about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days 14 days, or 15days after an antibody or fragment thereof, or composition comprisingthe antibody or functional fragment thereof. In another embodiment,compositions comprising apoptotic cell supernatants are administeredabout 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeksafter an antibody or functional fragment thereof, or compositioncomprising the antibody or functional fragment thereof.

In some embodiments, a composition comprising apoptotic cells isadministered independent of CAR T-cells. In some embodiments, acomposition comprising apoptotic cells is administered in combinationwith an additional agent. In some embodiments, the additional agent isan antibody.

In some embodiments, the composition as disclosed herein comprises atherapeutic composition. In some embodiments, the composition asdisclosed herein comprises a therapeutic efficacy.

In some embodiments, a composition as disclosed herein is administeredonce. In another embodiment, the composition is administered twice. Inanother embodiment, the composition is administered three times. Inanother embodiment, the composition is administered four times. Inanother embodiment, the composition is administered at least four times.In another embodiment, the composition is administered more than fourtimes.

In some embodiments, CAR T-cells as disclosed herein are administeredonce. In another embodiment, CAR T-cells are administered twice. Inanother embodiment, CAR T-cells are administered three times. In anotherembodiment, CAR T-cells are administered four times. In anotherembodiment, CAR T-cells are administered at least four times. In anotherembodiment, the composition is administered more than four times.

In some embodiments, the composition as disclosed herein is atherapeutic composition. In another embodiment, the composition asdisclosed herein has therapeutic efficacy.

In some embodiments, disclosed herein are a composition which providesreduced inflammatory cytokine or chemokine release compared to acomposition comprising CAR T-cells alone, but with comparablecytotoxicity compared to a composition comprising CAR T-cells alone.

Formulations

Pharmaceutical compositions disclosed herein comprising early apoptoticcell populations, can be conveniently provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may be buffered to aselected pH, Liquid preparations are normally easier to prepare thangels, other viscous compositions, and solid compositions. Additionally,liquid compositions are somewhat more convenient to administer,especially by injection. Viscous compositions, on the other hand, can beformulated within the appropriate viscosity range to provide longercontact periods with specific tissues. Liquid or viscous compositionscan comprise carriers, which can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol, and the like) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the earlyapoptotic cell population described herein and utilized in practicingthe methods disclosed herein, in the required amount of the appropriatesolvent with various amounts of the other ingredients, as desired. Suchcompositions may be in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can also be lyophilized. Thecompositions can contain auxiliary substances such as wetting,dispersing, or emulsifying agents (e.g., methylcellulose), pH bufferingagents, gelling or viscosity enhancing additives, preservatives,flavoring agents, colors, and the like, depending upon the route ofadministration and the preparation desired. Standard texts, such as“REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporatedherein by reference, may be consulted to prepare suitable preparations,without undue experimentation.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the disclosure herein,however, any vehicle, diluent, or additive used would have to becompatible with the genetically modified immunoresponsive cells or theirprogenitors.

The compositions or formulations described herein can be isotonic, i.e.,they can have the same osmotic pressure as blood and lacrimal fluid. Thedesired isotonicity of the compositions as disclosed herein may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride may be preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose may be preferred because it is readily and economicallyavailable and is easy to work with.

Other suitable thickening agents include, for example, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and thelike. The preferred concentration of the thickener will depend upon theagent selected. The important point is to use an amount that willachieve the selected viscosity. Obviously, the choice of suitablecarriers and other additives will depend on the exact route ofadministration and the nature of the particular dosage form, e.g.,liquid dosage form (e.g., whether the composition is to be formulatedinto a solution, a suspension, gel or another liquid form, such as atime release form or liquid-filled form).

Those skilled in the art will recognize that the components of thecompositions or formulations should be selected to be chemically inertand will not affect the viability or efficacy of the early apoptoticcell populations as described herein, for use in the methods disclosedherein. This will present no problem to those skilled in chemical andpharmaceutical principles, or problems can be readily avoided byreference to standard texts or by simple experiments (not involvingundue experimentation), from this disclosure and the documents citedherein.

One consideration concerning the therapeutic use of genetically modifiedimmunoresponsive cells disclosed herein is the quantity of cellsnecessary to achieve an optimal effect. The quantity of cells to beadministered will vary for the subject being treated. In a someembodiments, between 10⁴ to 10¹⁰, between 10⁵ to 10⁹, or between 10⁶ and10⁸ genetically modified immunoresponsive cells disclosed herein areadministered to a human subject. More effective cells may beadministered in even smaller numbers. In some embodiments, at leastabout 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, and 5×10⁸ genetically modifiedimmunoresponsive cells disclosed herein are administered to a humansubject. The precise determination of what would be considered aneffective dose may be based on factors individual to each subject,including their size, age, sex, weight, and condition of the particularsubject. Dosages can be readily ascertained by those skilled in the artfrom this disclosure and the knowledge in the art.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods disclosed herein. Typically, any additives (inaddition to the active cell(s) and/or agent(s)) are present in an amountof 0.001 to 50% (weight) solution in phosphate buffered saline, and theactive ingredient is present in the order of micrograms to milligrams,such as about 0.0001 to about 5 wt %. In another embodiment about 0.0001to about 1 wt %. In still another embodiment, about 0.0001 to about 0.05wt % or about 0.001 to about 20 wt %. In a further embodiment, about0.01 to about 10 wt %. In another embodiment, about 0.05 to about 5 wt%. Of course, for any composition to be administered to an animal orhuman, and for any particular method of administration, it is preferredto determine therefore: toxicity, such as by determining the lethal dose(LD) and LD50 in a suitable animal model e.g., rodent such as mouse;and, the dosage of the composition(s), concentration of componentstherein and timing of administering the composition(s), which elicit asuitable response. Such determinations do not require undueexperimentation from the knowledge of the skilled artisan, thisdisclosure and the documents cited herein. And, the time for sequentialadministrations can be ascertained without undue experimentation.

Nucleic Acid Sequences, Vectors, Cells

In some embodiments, disclosed herein are an isolated nucleic acidsequence encoding a chimeric antigen receptor (CAR) as described hereinfor uses in the compositions and methods as disclosed herein.

In another embodiment, disclosed herein are a vector comprising thenucleic acid sequence encoding a chimeric antigen receptor (CAR) asdescribed herein.

In some embodiments, disclosed herein are an isolated nucleic acidsequence encoding a genetically modified T-cell receptor (TCR) asdescribed herein for uses in the compositions and methods as disclosedherein. In another embodiment, disclosed herein are a vector comprisingthe nucleic acid sequence encoding a genetically modified T-cellreceptor (TCR) as described herein.

Genetic modification of immunoresponsive cells (e.g., T-cells, CTLcells, NK cells) can be accomplished by transducing a substantiallyhomogeneous cell composition with a recombinant DNA construct. In someembodiments, a retroviral vector (either gamma-retroviral or lentiviral)is employed for the introduction of the DNA construct into the cell. Forexample, a polynucleotide encoding a receptor that binds an antigen(e.g., a tumor antigen, or a valiant, or a fragment thereof), can becloned into a retroviral vector and expression can be driven from itsendogenous promoter, from the retroviral long terminal repeat, or from apromoter specific for a targeT-cell type of interest. Non-viral vectorsmay be used as well.

Non-viral approaches can also be employed for the expression of aprotein in cell. For example, a nucleic acid molecule can be introducedinto a cell by administering the nucleic acid in the presence oflipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413,1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al, Am.J. Med. Sci. 298:278, 1989; Staubinger et al, Methods in Enzymology101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al.,Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal ofBiological Chemistry 264:16985, 1989), or by micro-injection undersurgical conditions (Wolff et al., Science 247: 1465, 1990). Othernon-viral means for gene transfer include transfection in vitro usingcalcium phosphate, DEAE dextran, electroporation, and protoplast fusion.Liposomes can also be potentially beneficial for delivery of DNA into acell. Transplantation of normal genes into the affected tissues of asubject can also be accomplished by transferring a normal nucleic acidinto a cultivatable cell type ex vivo (e.g., an autologous orheterologous primary cell or progeny thereof), after which the cell (orits descendants) are injected into a targeted tissue or are injectedsystemically. Recombinant receptors can also be derived or obtainedusing transposases or targeted nucleases (e.g. Zinc finger nucleases,meganucleases, or TALE nucleases). Transient expression may be obtainedby RNA electroporation. cDNA expression for use in polynucleotidetherapy methods can be directed from any suitable promoter (e.g., thehuman cytomegalovirus (CMV), simian virus 40 (SV40), or metallothioneinpromoters), and regulated by any appropriate mammalian regulatoryelement or intron (e.g. the elongation factor laenhancer/promoter/intron structure). For example, if desired, enhancersknown to preferentially direct gene expression in specific cell typescan be used to direct the expression of a nucleic acid. The enhancersused can include, without limitation, those that are characterized astissue- or cell-specific enhancers. Alternatively, if a genomic clone isused as a therapeutic construct, regulation can be mediated by thecognate regulatory sequences or, if desired, by regulatory sequencesderived from a heterologous source, including any of the promoters orregulatory elements described above.

In another embodiment, disclosed herein are a cell comprising the vectorcomprising the nucleic acid sequence encoding a chimeric antigenreceptor (CAR) as disclosed herein.

Methods of Use

In one embodiment, disclosed herein are methods for treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor comprising the step of administering acomposition as disclosed herein.

In some embodiments, disclosed herein are methods of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor in a subject comprising the step ofadministering a composition comprising apoptotic cells. In someembodiments, disclosed herein are methods of treating, preventing,inhibiting the growth of, delaying disease progression, reducing thetumor load, or reducing the incidence of a cancer or a tumor in asubject, or any combination thereof. In some embodiments, methodsdisclosed herein reduce the size and or growth rate of a tumor orcancer. In some embodiments, methods disclosed herein increase thesurvival of a subject suffering from a tumor or cancer. In someembodiments, use of apoptotic cells or a composition thereof increasesthe efficacy of genetically modified immune cell therapy, for examplebut not limited to CAR T-cell therapy.

In another embodiment, disclosed herein are methods for treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor comprising the step of administeringgenetically modified immune cells and a composition comprising anadditional agent, wherein said additional agent comprises apoptoticcells, a supernatant from apoptotic cells, a CTLA-4 blocking agent, analpha-1 anti-trypsin or fragment or analog thereof, a tellurium-basedcompound, or an immune modulating agent, or any combination thereof,wherein said method treats, prevents, inhibits, reduces the incidenceof, ameliorates or alleviates a cancer or a tumor in said subjectcompared with a subject administered said genetically modified immunecells and not administered the additional agent. In another embodiment,said genetically modified immune cells comprise genetically modifiedT-cell, cytotoxic T-cells, Treg cells, effector T-cells, helper T-cells,NK cells, or dendritic cells.

In another embodiment, disclosed herein are methods for treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor comprising the step of administeringchimeric antigen receptor-expressing T-cells (CAR T-cells) and acomposition comprising an additional agent, wherein said additionalagent comprises apoptotic cells, a supernatant from apoptotic cells, aCTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment or analogthereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof, wherein said method treats, prevents, inhibits,reduces the incidence of, ameliorates or alleviates a cancer or a tumorin said subject compared with a subject administered said geneticallymodified immune cells and not administered the additional agent.

In another embodiment, disclosed herein are methods for treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor comprising the step of administeringgenetically modified T-cell receptor cells (TCR T-cells) and acomposition comprising an additional agent, wherein said additionalagent comprises apoptotic cells, a supernatant from apoptotic cells, aCTLA-4 blocking agent, an alpha-1 anti-trypsin or fragment or analogthereof, a tellurium-based compound, or an immune modulating agent, orany combination thereof, wherein said method treats, prevents, inhibits,reduces the incidence of, ameliorates or alleviates a cancer or a tumorin said subject compared with a subject administered said geneticallymodified immune cells and not administered the additional agent.

In another embodiment, administration of apoptotic cells or an apoptoticsupernatant or compositions thereof does not reduce the efficacy fortreating, preventing, inhibiting, reducing the incidence of,ameliorating, or alleviating a cancer or a tumor, of said administeringchimeric antigen receptor-expressing T-cells. In another embodiment,administration of an additional agent comprising apoptotic cells, asupernatant from apoptotic cells, a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment or analog thereof, a tellurium-based compound,or an immune modulating agent, or any combination thereof, orcompositions thereof does not reduce the efficacy for treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor, of said administering chimeric antigenreceptor-expressing T-cells.

In another embodiment, administration of apoptotic cells or an apoptoticsupernatant or compositions thereof increases the efficacy for treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor, of said administering chimeric antigenreceptor-expressing T-cells. In another embodiment, administration of anadditional agent comprising apoptotic cells, a supernatant fromapoptotic cells, a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment or analog thereof, a tellurium-based compound, or an immunemodulating agent, or any combination thereof, or compositions thereofincreases the efficacy for treating, preventing, inhibiting, reducingthe incidence of, ameliorating, or alleviating a cancer or a tumor, ofsaid administering chimeric antigen receptor-expressing T-cells.

In one embodiment, methods increasing the efficacy of a geneticallymodified immune cell cancer therapy comprise administering saidgenetically modified immune cells and an additional agent comprisingapoptotic cells, a supernatant from apoptotic cells, a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment or analog thereof, atellurium-based compound, or an immune modulating agent, or anycombination thereof, or compositions thereof, wherein the efficacy isincreased compared with a subject not administered said additionalagent. In another embodiment said genetically modified immune cells areT-cells. In another embodiment, a T-cell is a naïve T-cell. In anotherembodiment, a T-cell is a naïve CD4⁺ T-cell. In another embodiment, aT-cell is a naïve T-cell. In another embodiment, a T-cell is a naïveCD8⁺ T-cell. In another embodiment, the genetically modified immune cellis a natural killer (NK) cell. In another embodiment, the geneticallymodified immune cell is a dendritic cell. In still another embodiment,the genetically modified T-cell is a cytotoxic T lymphocyte (CTL cell).In another embodiment, the genetically modified T-cell is a regulatoryT-cell (Treg). In another embodiment, the genetically modified T-cell isa chimeric antigen receptor (CAR) T-cell. In another embodiment, thegenetically modified T-cell is a genetically modified T-cell receptorcell (TCR T-cell). In another embodiment, methods increasing theefficacy of a CAR T-cell cancer therapy comprise administering saidgenetically modified immune cells and an additional agent comprisingapoptotic cells, a supernatant from apoptotic cells, a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment or analog thereof, atellurium-based compound, or an immune modulating agent, or anycombination thereof, or compositions thereof, wherein the efficacy isincreased compared with a subject not administered said additionalagent.

In another embodiment, methods herein reduce the level of production ofat least one pro-inflammatory cytokine compared with the level of saidpro-inflammatory cytokine in a subject receiving an immune cancertherapy and not administered an additional agent. In another embodiment,methods herein inhibit or reduce the incidence of cytokine releasesyndrome or cytokine storm in a subject undergoing a geneticallymodified immune cell cancer therapy and not administered an additionalagent.

In another embodiment, methods disclosed herein reduce IL-6.

In another embodiment, methods herein increase the production of atleast one cytokine compared with the level of said cytokine in a subjectreceiving an immune cancer therapy and not administered an additionalagent. In some embodiments, the additional agent is apoptotic cells, Inother embodiment, the additional agent is an apoptotic cell supernatant.In another embodiment, methods disclosed herein increase IL-2.

A skilled artisan would appreciate that the term “production” as usedherein in reference to a cytokine, may encompass expression of thecytokine as well as secretion of the cytokine from a cell. In oneembodiment, increased production of a cytokine results in increasedsecretion of the cytokine from the cell. In an alternate embodiment,decreased production of a cytokine results in decreased secretion of thecytokine from the cell. In another embodiment, methods disclosed hereindecrease secretion of at least one cytokine. In another embodiment,methods disclosed herein decrease secretion of IL-6. In anotherembodiment, methods disclosed herein increase secretion of at least onecytokine. In another embodiment, methods disclosed herein increasesecretion of IL-2.

In some embodiments, the methods disclosed herein modulate theproduction or level of one or more factors in a treated subject, forexample but not limited to, as herein exemplified in Examples 19 and 20below. In certain embodiments, the method decreases the production orlevel of C-reactive protein (CRP). In certain embodiments, the methoddecreases the production or level of IL-6. In certain embodiments, themethod decreases the production or level of TNF-α. In certainembodiments, the method decreases the production or level of IL-1β. Incertain embodiments, the method decreases the production or level ofIL-18. In certain embodiments, the method decreases the production orlevel of IFN-γ. In certain embodiments, the method decreases theproduction or level of IL-10. In certain embodiments, the methoddecreases the production or level of IL-1Ra. In certain embodiments, themethod decreases the production or level of TNFR-1. In certainembodiments, the method decreases the production or level of G-CSF. Incertain embodiments, the method decreases the production or level ofVEGF. In certain embodiments, the method decreases the production orlevel of GM-CSF. In certain embodiments, the method decreases theproduction or level of MCP-1. In certain embodiments, the methoddecreases the production or level of IP-10. In certain embodiments, themethod decreases the production or level of MIP-1α. In certainembodiments, the method decreases the production or level of IL-8. Incertain embodiments, the method increases the production or level ofGro-β. In certain embodiments, the method increases the production orlevel of RANTES. In certain embodiments, the method decreases theproduction or level of TRAM-1. In certain embodiments, the methoddecreases the production or level of Osteopontin. In certainembodiments, the method decreases the production or level of NGAL. Incertain embodiments, the method increases the production or level ofGhrelin. In certain embodiments, the method decreases the production orlevel of leptin. In certain embodiments, the method decreases theproduction or level of glucagon. In certain embodiments, the methoddecreases the production or level of cortisol. In certain embodiments,the method increases the production or level of FT3.

In another embodiment, a cell secreting at least one cytokine is a tumorcell. In another embodiment, a cell secreting at least one cytokine is aT-cell. In another embodiment, a cell secreting at least one cytokine isan immune cell. In another embodiment, a cell secreting at least onecytokine is a macrophage. In another embodiment, a cell secreting atleast one cytokine is a B cell lymphocyte. In another embodiment, a cellsecreting at least one cytokine is a mast cell. In another embodiment, acell secreting at least one cytokine is an endothelial cell. In anotherembodiment, a cell secreting at least one cytokine is a fibroblast. Inanother embodiment, a cell secreting at least one cytokine is a stromalcell. A skilled artisan would recognize that the level of cytokines maybe increased or decreased in cytokine secreting cells depending on theenvironment surrounding the cell.

In yet another embodiment, an additional agent used in the methodsdisclosed herein increases secretion of at least one cytokine. In yetanother embodiment, an additional agent used in the methods disclosedherein maintains secretion of at least one cytokine. In still anotherembodiment, an additional agent used in the methods disclosed hereindoes not decrease secretion of at least one cytokine. In anotherembodiment, an additional agent used in the methods disclosed hereinincreases secretion of IL-2. In another embodiment, an additional agentused in the methods disclosed herein increases secretion of IL-2R. Inanother embodiment, an additional agent used in the methods disclosedherein maintains secretion levels of IL-2. In another embodiment, anadditional agent used in the methods disclosed herein maintainssecretion levels of IL-2R. In another embodiment, an additional agentused in the methods disclosed herein does not decrease secretion levelsof IL-2R. In another embodiment, an additional agent used in the methodsdisclosed herein maintains or increases secretion levels of IL-2. Inanother embodiment, an additional agent used in the methods disclosedherein maintains or increases secretion levels of IL-2R. In anotherembodiment, an additional agent used in the methods disclosed hereindoes not decrease secretion levels of IL-2R.

In still a further embodiment, an additional agent used in the methodsdisclosed herein decreases secretion of IL-6. In another embodiment, anadditional agent used in the methods disclosed herein maintains,increases, or does not decrease secretion levels of IL-2 whiledecreasing secretion of IL-6. In another embodiment, an additional agentused in the methods disclosed herein maintains, increases, or does notdecrease secretion levels of IL-2R while decreasing secretion of IL-6.

In one embodiment, methods of increasing the efficacy of a CAR T-cellcancer therapy disclosed herein comprises decreasing the level of IL-6in said subject, said method comprising administering CAR T-cells and anadditional agent comprising apoptotic cells, a supernatant fromapoptotic cells, a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment or analog thereof, a tellurium-based compound, or an immunemodulating agent, or any combination thereof, or compositions thereof,wherein the efficacy is increased compared with a subject notadministered said additional agent. In another embodiment, methods ofincreasing the efficacy of a CAR T-cell cancer therapy disclosed hereincomprises increasing the level of IL-2 in said subject, said methodcomprising administering CAR T-cells and an additional agent comprisingapoptotic cells, a supernatant from apoptotic cells, a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment or analog thereof, atellurium-based compound, or an immune modulating agent, or anycombination thereof, or compositions thereof, wherein the efficacy isincreased compared with a subject not administered said additionalagent. In another embodiment, methods of increasing the efficacy of aCAR T-cell cancer therapy disclosed herein comprises increasingproliferation of said CAR T-cells, said method comprising administeringCAR T-cells and an additional agent comprising apoptotic cells, asupernatant from apoptotic cells, a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment or analog thereof, a tellurium-based compound,or an immune modulating agent, or any combination thereof, orcompositions thereof, wherein the efficacy and proliferation of said CART-cells is increased compared with a subject not administered saidadditional agent.

In one embodiment, methods of increasing the efficacy of CAR T-cellcancer therapy, decrease or inhibit cytokine production in the subject,said methods comprising the step of administering a compositioncomprising CAR T-cells and a CTLA-4 blocking agent, an alpha-1anti-trypsin or fragment or analog thereof, a tellurium-based compound,or an immune modulating agent, or any combination thereof, orcompositions thereof. In another embodiment, methods of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or tumor also decrease or inhibit cytokineproduction in the subject, said methods comprising the step ofadministering a composition comprising CAR T-cells and a CTLA-4 blockingagent, an alpha-1 anti-trypsin or fragment or analog thereof, atellurium-based compound, or an immune modulating agent, or anycombination thereof, or compositions thereof.

In another embodiment, disclosed herein are methods of treating cytokinerelease syndrome or cytokine storm in a subject undergoing CAR T-cellcancer therapy.

In another embodiment, methods of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating a cancer ortumor, decrease or inhibit cytokine production in a subject, saidmethods comprising the step of administering a composition comprisingCAR T-cells and a CTLA-4 blocking agent, an alpha-1 anti-trypsin orfragment or analog thereof, a tellurium-based compound, or an immunemodulating agent, or any combination thereof, or compositions thereof.

In another embodiment, disclosed herein is a method of treating a canceror a tumor in a subject, said method comprising the step ofadministering to said subject any of the compositions as describedherein. In another embodiment, disclosed herein is a method ofpreventing a cancer or a tumor in a subject, said method comprising thestep of administering to said subject any of the compositions asdescribed herein. In another embodiment, disclosed herein is a method ofinhibiting a cancer or a tumor in a subject, said method comprising thestep of administering to said subject any of the compositions asdescribed herein. In another embodiment, disclosed herein is a method ofreducing a cancer or a tumor in a subject, said method comprising thestep of administering to said subject any of the compositions asdescribed herein. In another embodiment, disclosed herein is a method ofameliorating a cancer or a tumor in a subject, said method comprisingthe step of administering to said subject any of the compositions asdescribed herein. In another embodiment, disclosed herein is a method ofalleviating a cancer or a tumor in a subject, said method comprising thestep of administering to said subject any of the compositions asdescribed herein.

In one embodiment, disclosed herein are methods of maintaining orincreasing the proliferation rate of a genetically modified immune cellduring an immunotherapy, the method comprising the step of administeringa composition comprising apoptotic cells or an apoptotic supernatantduring the immunotherapy. In another embodiment, said geneticallymodified immune cells comprise a T-cell, a naïve T-cell, a naïve CD4⁺T-cell, a naïve CD8⁺ T-cell, a natural killer (NK) cell, a dendriticcell, a cytotoxic T lymphocyte (CTL cell), a regulatory T-cell (Treg), achimeric antigen receptor (CAR) T-cell, or a genetically modified T-cellreceptor (TCR) cell. In another embodiment, disclosed herein are methodsof maintaining or increasing the proliferation rate of a CAR T-cellduring an immunotherapy, the method comprising the step of administeringa composition comprising apoptotic cells or an apoptotic supernatantduring the immunotherapy.

In another embodiment, methods of maintaining or increasing theproliferation rate of the genetically modified immune cells does notreduce or inhibit the efficacy of the immunotherapy. For example, inanother embodiment, methods of maintaining or increasing theproliferation rate of CAR T-cells does not reduce or inhibit theefficacy of the CAR T-cell cancer therapy. In another embodiment,methods of maintaining or increasing the proliferation rate of thegenetically modified immune cells, for example CAR T-cells, decrease orinhibit cytokine production in the subject.

In another embodiment, compositions and methods as disclosed hereinutilize combination therapy with apoptotic cells or apoptoticsupernatants as disclosed herein, and one or more CTLA-4-blocking agentssuch as Ipilimumab. In one embodiment, CTLA-4 is a potent inhibitor ofT-cell activation that helps to maintain self-tolerance. In oneembodiment, administration of an anti-CTLA-4 blocking agent, which inanother embodiment, is an antibody, produces a net effect of T-cellactivation. In another embodiment, compositions and methods as disclosedherein utilize combined therapy comprising apoptotic cells, CAR T-cells,and one or more CTLA-4-blocking agents.

In some cases, a polypeptide of and for use in the methods as disclosedherein comprises at least one conservative amino acid substitutionrelative to an unmodified amino acid sequence. In other cases, thepolypeptide comprises a non-conservative amino acid substitution. Insuch cases, polypeptides having such modifications exhibit increasedstability or a longer half-life relative to a polypeptide lacking suchan amino acid substitution.

In some embodiment, “treating” comprises therapeutic treatment and“preventing” comprises prophylactic or preventative measures, whereinthe object is to prevent or lessen the targeted pathologic condition ordisorder as described hereinabove. Thus, in some embodiments, treatingmay include directly affecting or curing, suppressing, inhibiting,preventing, reducing the severity of, delaying the onset of, reducingsymptoms associated with the disease, disorder or condition, or acombination thereof. Thus, in some embodiments, “treating,”“ameliorating,” and “alleviating” refer inter alia to delayingprogression, expediting remission, inducing remission, augmentingremission, speeding recovery, increasing efficacy of or decreasingresistance to alternative therapeutics, or a combination thereof. Insome embodiments, “preventing” refers, inter alia, to delaying the onsetof symptoms, preventing relapse to a disease, decreasing the number orfrequency of relapse episodes, increasing latency between symptomaticepisodes, or a combination thereof. In some embodiments, “suppressing”or “inhibiting”, refers inter alia to reducing the severity of symptoms,reducing the severity of an acute episode, reducing the number ofsymptoms, reducing the incidence of disease-related symptoms, reducingthe latency of symptoms, ameliorating symptoms, reducing secondarysymptoms, reducing secondary infections, prolonging patient survival, ora combination thereof.

A skilled artisan would appreciate that the term “antigen recognizingreceptor” may encompass a receptor that is capable of activating animmune cell (e.g., a T-cell) in response to antigen binding. Exemplaryantigen recognizing receptors may be native or endogenous T-cellreceptors or chimeric antigen receptors in which a tumor antigen-bindingdomain is fused to an intracellular signaling domain capable ofactivating an immune cell (e.g., a T-cell).

In some embodiments, methods described herein increase the survival of asubject suffering from a cancer or a tumor, and comprise administeringan early apoptotic cell population to said subject, wherein the methodincreases the survival of the subject.

A skilled artisan would appreciate that the term “disease” may encompassany condition or disorder that damages or interferes with the normalfunction of a cell, tissue, or organ. Examples of diseases includeneoplasia or pathogen infection of cell.

A skilled artisan would appreciate that the term “effective amount” mayencompass an amount sufficient to have a therapeutic effect. In someembodiments, an “effective amount” is an amount sufficient to arrest,ameliorate, or inhibit the continued proliferation, growth, ormetastasis (e.g., invasion, or migration) of a neoplasia.

A skilled artisan would appreciate that the term “neoplasia” mayencompass a disease characterized by the pathological proliferation of acell or tissue and its subsequent migration to or invasion of othertissues or organs. Neoplasia growth is typically uncontrolled andprogressive, and occurs under conditions that would not elicit, or wouldcause cessation of, multiplication of normal cells. Neoplasias canaffect a variety of cell types, tissues, or organs, including but notlimited to an organ selected from the group consisting of bladder, bone,brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder,heart, intestines, kidney, liver, lung, lymph node, nervous tissue,ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen,stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter,urethra, uterus, and vagina, or a tissue or cell type thereof.Neoplasias include cancers, such as sarcomas, carcinomas, orplasmacytomas (malignant tumor of the plasma cells).

A skilled artisan would appreciate that the term “pathogen” mayencompass a virus, bacteria, fungi, parasite or protozoa capable ofcausing disease.

A skilled artisan would appreciate that the term “tumor antigen” or“tumor associated antigen” may encompass an antigen (e.g., apolypeptide) that is uniquely or differentially expressed on a tumorcell compared to a normal or non-IS neoplastic cell. With reference tothe compositions and methods disclosed herein, a tumor antigen includesany polypeptide expressed by a tumor that is capable of activating orinducing an immune response via an antigen recognizing receptor (e.g.,CD 19, MUCI) or capable of suppressing an immune response viareceptor-ligand binding (e.g., CD47, PD-L1/L2, B7.1/2).

A skilled artisan would appreciate that the term “virus antigen” mayencompass a polypeptide expressed by a virus that is capable of inducingan immune response.

The terms “comprises”, “comprising”, and are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like. Similarly, the term “consists of” and“consists essentially of” have the meanings ascribed to them in U.S.Patent Law. The compositions and methods as disclosed herein areenvisioned to either comprise the active ingredient or specified step,consist of the active ingredient or specified step, or consistessentially of the active ingredient or specified step.

A skilled artisan would appreciate that the term “treatment” mayencompass clinical intervention in an attempt to alter the diseasecourse of the individual or cell being treated, and can be performedeither for prophylaxis or during the course of clinical pathology.Therapeutic effects of treatment include, without limitation, preventingoccurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastases, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. By preventing progression of a diseaseor disorder, a treatment can prevent deterioration due to a disorder inan affected or diagnosed subject or a subject suspected of having thedisorder, but also a treatment may prevent the onset of the disorder ora symptom of the disorder in a subject at risk for the disorder orsuspected of having the disorder.

A skilled artisan would appreciate that the term “subject” may encompassa vertebrate, in some embodiments, to a mammal, and in some embodiments,to a human. Subject may also refer, in some embodiments, to domesticatedsuch as cows, sheep, horses, cats, dogs and laboratory animals such asmice, rats, gerbils, hamsters, etc.

In some embodiments, disclosed herein are CAR T-cells in which the CARis directed to a peptide of interest. In some embodiments, the CAR bindsto a peptide of interest. In another embodiment, the CAR targets apeptide of interest. In another embodiment, the CAR activates a peptideof interest. In another embodiment, the CAR is a ligand of the peptideof interest. In another embodiment, the peptide of interest is a ligandof the CAR. Each of these embodiments is to be considered part disclosedherein.

In some embodiments, the immune cell as disclosed herein is not aT-cell. In another embodiment, the immune cell as disclosed herein isnot an NK cell. In another embodiment, the immune cell as disclosedherein is not a CTL. In another embodiment, the immune cell as disclosedherein is not a regulatory T-cell. In another embodiment, the immunecell is not a human embryonic stem cell. In another embodiment, theimmune cell is not a pluripotent stem cell from which lymphoid cells maybe differentiated.

One approach to immunotherapy involves engineering a patient's ownimmune cells to create genetically modified immune cells that willrecognize and attack their tumor. Immune cells are collected andgenetically modified, as described herein, for example to producechimeric antigen receptors (CAR) on their cell surface that will allowthe immune cell, for example a T-cell, to recognize a specific proteinantigen on a tumor or cancer cell. An expanded population of geneticallymodified immune cells, for example CAR T-cells, is then administered tothe patient. In some embodiments, the administered cells multiply in thepatient's body and recognize and kill cancer and tumor cells that harborthe antigen on their surface. In another embodiment, the administeredcells multiply in a patient's body and recognize and killtumor-associated antigens, which leads to the death of cancer and tumorcells.

In some embodiments, disclosed herein are methods of inhibiting orreducing the incidence of cytokine release syndrome or cytokine storm ina subject undergoing CAR T-cell cancer therapy, and methods ofdecreasing or inhibiting cytokine production in a subject experiencingcytokine release syndrome or cytokine storm, said methods comprising thestep of administering a composition comprising apoptotic cells or asupernatant of apoptotic cells. In another embodiment, disclosed hereinare methods of treating cytokine release syndrome or cytokine storm in asubject undergoing CAR T-cell cancer therapy. In another embodiment,disclosed herein are methods of preventing cytokine release syndrome orcytokine storm in a subject undergoing CAR T-cell cancer therapy. Inanother embodiment, disclosed herein are methods of alleviating cytokinerelease syndrome or cytokine storm in a subject undergoing CAR T-cellcancer therapy. In another embodiment, disclosed herein are methods ofameliorating cytokine release syndrome or cytokine storm in a subjectundergoing CAR T-cell cancer therapy. In another embodiment,administration of apoptotic cells or an apoptotic supernatant orcompositions thereof does not reduce the efficacy of the CAR T-celltherapy.

In some embodiments, disclosed herein are methods of inhibiting orreducing the incidence of a cytokine release syndrome (CRS) or acytokine storm in a subject undergoing chimeric antigenreceptor-expressing T-cell (CAR T-cell) cancer therapy, wherein themethod comprises the step of administering a composition comprisingapoptotic cells or an apoptotic cell supernatant or compositions thereofto said subject. In another embodiment, inhibiting or reducing theincidence of a cytokine release syndrome (CRS) or a cytokine storm isdetermined by measuring cytokine levels in a subject undergoing chimericantigen receptor-expressing T-cell cancer therapy and being administeredapoptotic cells or an apoptotic supernatant. In another embodiment,measured levels of cytokines are compared with cytokine levels in asubject not undergoing CAR T-cell cancer therapy. In another embodiment,measured cytokine levels are compared with cytokine levels in a subjectnot administer apoptotic cells or an apoptotic supernatant. In yetanother embodiment, measured cytokine levels are compared with a controlsubject.

In another embodiment, the level of pro-inflammatory cytokines arereduced in the subject compared with a subject undergoing CAR T-cellcancer therapy and not administered said apoptotic cells or saidapoptotic cell supernatant or compositions thereof. In anotherembodiment, methods disclosed herein reduce or inhibit the level ofproduction of at least one pro-inflammatory cytokines compared with asubject undergoing CAR T-cell cancer therapy and not administered saidapoptotic cells or said apoptotic cell supernatant or compositionsthereof.

In another embodiment, a method disclosed herein may further compriseadministration of additional agents. Alternatively, a method disclosedherein may comprise administration of additional agents and notapoptotic cells or an apoptotic cell supernatant. In still a furtherembodiment, additional agents may be those compounds or compositionsthat enhance or improve, or any combination thereof, CAR T-cell cancertherapy. In yet a further embodiment, additional agents that enhance orimprove CAR T-cell cancer therapy include CTLA-4 blocking agents, analpha-lanti-trypsin or functional fragment thereof, or an analoguethereof, a tellurium-based compound, or an immune-modulating agent, orany combination thereof. In another embodiment, an additional agentincludes apoptotic cells or an apoptotic supernatant. In anotherembodiment, administration of an additional agent, a described herein,is prior to, concurrent with, of following said CAR T-cell cancertherapy.

In some embodiments, an IL-6 receptor antagonist, which in oneembodiment is tocilizumab is used with the compositions and methods asdisclosed herein.

In some embodiments, adoptively transferred T-cells engraft and expandmore efficiently in a lymphopenic host. Thus, in some embodiments, thesubject is subjected to lymphodepletion prior to transfer of CAR T-cellsor other modified immune cells. In another embodiment, the subjectreceiving the CAR T-cells is given T-cell-supportive cytokines.

In some embodiments, the T-cells are effector T-cells. In anotherembodiment, the T-cells are naïve T-cells. In another embodiment, theT-cells are central memory (T_(CM)) T-cells. In another embodiment, theT-cells are Th17 cells. In another embodiment, the T-cells are T stemmemory cells. In another embodiment, the T-cells have high replicativecapacity. In another embodiment, T-cell expansion occurs in the patient.In another embodiment, small numbers of cells may be transferred to apatient. In another embodiment, T-cell expansion occurs in vitro. Inanother embodiment, large numbers of cells may be transferred to apatient, cells and/or supernatants may be transferred to a patient onmultiple occasions, or a combination thereof.

In some embodiments, an advantage of CAR T-cells is that because theyare specific for cell-surface molecules, they overcome the constraintsof MHC-restricted TCR recognition and avoid tumor escape throughimpairments in antigen presentation or human leukocyte antigenexpression.

In some embodiments, disclosed herein is a method of reducing a tumorburden in a subject, said method comprising the step of administering tosaid subject any of the compositions as described herein.

In some embodiments, reducing the tumor burden comprises reducing thenumber of tumor cells in the subject. In another embodiment, reducingthe tumor burden comprises reducing tumor size in the subject. Inanother embodiment, reducing the tumor burden comprises eradicating thetumor in the subject.

In another embodiment, disclosed herein is a method of inducing tumorcell death in a subject, said method comprising the step ofadministering to said subject any of the compositions as describedherein. In another embodiment, a method as disclosed herein for inducingtumor cell death in a subject comprises administering immune cells, suchas NK cells or T-cells comprising engineered chimeric antigen receptorswith at least an additional agent to decrease toxic cytokine release or“cytokine release syndrome” (CRS) or “severe cytokine release syndrome”(sCRS) or “cytokine storm” in the subject.

In another embodiment, disclosed herein is a method of increasing orlengthening the survival of a subject having neoplasia, comprising thestep of administering to said subject any of the compositions asdescribed herein. In another embodiment, a method of increasing orlengthening the survival of a subject comprises administering immunecells, such as NK cells or T-cells comprising engineered chimericantigen receptors with at least an additional agent to decrease toxiccytokine release or “cytokine release syndrome” (CRS) or “severecytokine release syndrome” (sCRS) or “cytokine storm” in the subject.

In another embodiment, disclosed herein is a method of increasing orlengthening the survival of a subject having neoplasia, comprising thestep of administering to said subject any of the compositions asdescribed herein.

In some embodiments, disclosed herein is a method of delaying cancerprogression in a subject, comprising a step of administering to thesubject any of the compositions or combinations of compositionsdescribed herein. In some embodiments, disclosed herein is a method ofdelaying progression of a leukemia or lymphoma in a subject, comprisinga step of administering to the subject any of the compositions orcombinations of compositions described herein. In some embodiments,disclosed herein is a method of increasing, extending, or prolonging thesurvival of a subject suffering from a cancer or a tumor, comprising astep of administering to the subject any of the compositions orcombinations of compositions described herein. In some embodiments,disclosed herein is a method of increasing, extending, or prolonging thesurvival of a subject suffering from a leukemia or lymphoma, comprisingadministering to the subject any of the compositions or combinations ofcompositions described herein. In some embodiments, disclosed herein isa method of reducing the tumor cell burden in a subject, comprisingadministering to the subject any of the compositions or combinations ofcompositions described herein. In some embodiments, tumor burden isreduced the liver and bone marrow.

In another embodiment, disclosed herein is a method of preventingneoplasia in a subject, said method comprising the step of administeringto the subject any of the compositions or combinations of compositionsdescribed herein. In some embodiments, the neoplasia is selected fromthe group consisting of blood cancer, B cell leukemia, multiple myeloma,lymphoblastic leukemia (ALL), chronic lymphocytic leukemia,non-Hodgkin's lymphoma, ovarian cancer, or a combination thereof.

In another embodiment, disclosed herein is a method of treating bloodcancer in a subject in need thereof, comprising the step ofadministering to said subject any of the compositions as describedherein. In some embodiments, the blood cancer is selected from the groupconsisting of B cell leukemia, multiple myeloma, acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia, and non-Hodgkin'slymphoma.

In some embodiments, administration comprises administering acomposition comprising CAR T-cells. In some embodiments, administrationcomprises administering a composition comprising early apoptotic cells.In some embodiments, administration comprises administering acomposition comprising a supernatant obtained from early apoptoticcells. In some embodiments, administration comprises administering acombination of compositions described herein. In some embodiments,administration comprises administering CAR T-cells and apoptotic cellsin the same or different compositions. In some embodiments,administration comprises administering CAR T-cells in combination withan additional agent as described herein. In some embodiments,administration comprises administering apoptotic cells and an antibodyor fragment thereof in the same or different compositions.

In some embodiments, combination therapy provides a synergistic effect.In some embodiments, methods of use an early apoptotic cells incombination with CAR T-cells increases CAR T-cell efficacy in comparisonto use of CAR T-cells alone. In some embodiments, methods of use anearly apoptotic cells in combination with CAR T-cells extends thesurvival time of a subject suffering from a cancer or tumor incomparison to use of CAR T-cells alone. In some embodiments, methods ofuse an early apoptotic cells in combination with CAR T-cells extends thesurvival time of a subject suffering from a lymphoma or leukemia incomparison to use of CAR T-cells alone.

In some embodiments, methods of use an early apoptotic cells incombination with an antibody or fragment thereof delays the onset ofcancer or the appearance of a tumor, in comparison to use of eitherapoptotic cells or the antibody alone. In some embodiments, methods ofuse an early apoptotic cells in combination with an antibody or fragmentthereof delays the progression of a cancer, in comparison to use ofeither apoptotic cells or the antibody alone. In some embodiments,methods of use an early apoptotic cells in combination with an antibodyor fragment thereof delays the growth of a tumor, in comparison to useof either apoptotic cells or the antibody alone. In some embodiments,methods of use an early apoptotic cells in combination with an antibodyor fragment thereof extends the survival time of a subject sufferingfrom a cancer or tumor in comparison to use of either apoptotic cells orthe antibody alone. In some embodiments, methods of use an earlyapoptotic cells in combination with an antibody or fragment thereofextends the survival time of a subject suffering from a lymphoma orleukemia in comparison to use of either apoptotic cells or the antibodyalone.

In some embodiments, methods of use comprising administration of anearly apoptotic cells in combination with an antibody or fragmentthereof comprising RtX, delays the onset of cancer or the appearance ofa tumor, in comparison to use of either apoptotic cells or the antibodyalone. In some embodiments, methods of use an early apoptotic cells incombination with an antibody or fragment thereof comprising RtX, delaysthe progression of a cancer, in comparison to use of either apoptoticcells or the antibody alone. In some embodiments, methods of use anearly apoptotic cells in combination with an antibody or fragmentthereof comprising RtX, delays the growth of a tumor, in comparison touse of either apoptotic cells or the antibody alone. In someembodiments, methods of use an early apoptotic cells in combination withan antibody or fragment thereof comprising RtX, extends the survivaltime of a subject suffering from a cancer or tumor in comparison to useof either apoptotic cells or the antibody alone. In some embodiments,methods of use an early apoptotic cells in combination with an antibodyor fragment thereof comprising RtX, extends the survival time of asubject suffering from a lymphoma or leukemia in comparison to use ofeither apoptotic cells or the antibody alone.

In some embodiments, methods of use described herein reduce tumor load.A skilled artisan would appreciate that the term “tumor load” may referto the number of cancer cells, the size of a tumor, or the amount ofcancer in the body. The term “tumor load” may be used interchangeablywith the term “tumor burden” having all the same meanings and qualities.In some embodiments, methods of use comprising administration of anearly apoptotic cells reduces the number of cancer cells in a subject,reduces the size of a tumor in a subject, or reduces the amount ofcancer in the body of a subject, or any combination thereof comparedwith a subject not administered apoptotic cells. In some embodiments,methods of use comprising administration of an early apoptotic cells incombination with an antibody or fragment thereof reduces the number ofcancer cells in a subject, reduces the size of a tumor in a subject, orreduces the amount of cancer in the body of a subject, or anycombination thereof, compared with a subject not administered apoptoticcells or not administer the antibody, or the combination thereof. Insome embodiments, methods of use comprising administration of an earlyapoptotic cells in combination with a RtX antibody or fragment thereofreduces the number of cancer cells in a subject, reduces the size of atumor in a subject, or reduces the amount of cancer in the body of asubject, or any combination thereof, compared with a subject notadministered apoptotic cells, the RtX antibody, or the combinationthereof.

In some embodiments, a method of decreasing or inhibiting cytokineproduction in a subject experiencing cytokine release syndrome orcytokine storm or vulnerable to a cytokine release syndrome or cytokinestorm, as disclosed herein, decreases or inhibits cytokine production.In another embodiment, the method decreases or inhibits pro-inflammatorycytokine production. In a further embodiment, the method decreases orinhibits at least one pro-inflammatory cytokine. In another embodiment,wherein the subject is undergoing CAR T-cell cancer therapy, the methoddoes not reduce the efficacy of the CAR T-cell therapy.

The methods provided herein comprise administering a T-cell, NK cell, orCTL cell disclosed herein, in in an amount effective to achieve thedesired effect, be it palliation of an existing condition or preventionof recurrence. For treatment, the amount administered is an amounteffective in producing the desired effect. An effective amount can beprovided in one or a series of administrations. An effective amount canbe provided in a bolus or by continuous perfusion.

A skilled artisan would recognize that an “effective amount” (or,“therapeutically effective amount”) may encompass an amount sufficientto effect a beneficial or desired clinical result upon treatment. Aneffective amount can be administered to a subject in one or more doses.In terms of treatment, an effective amount is an amount that issufficient to palliate, ameliorate, stabilize, reverse or slow theprogression of the disease, or otherwise reduce the pathologicalconsequences of the disease. The effective amount is generallydetermined by the physician on a case-by-case basis and is within theskill of one in the art. Several factors are typically taken intoaccount when determining an appropriate dosage to achieve an effectiveamount. These factors include age, sex and weight of the subject, thecondition being treated, the severity of the condition and the form andeffective concentration of the antigen-binding fragment administered.

In one embodiment, methods disclosed herein comprise administering acomposition comprising a genetically modified cell, and the additionalagent or combination thereof, comprised in a single composition. Inanother embodiment, methods comprise administering a compositioncomprising a CAR T-cell, and the additional agent or combinationthereof, comprised in a single composition. In another embodiment,methods comprise administering a composition comprising a TCR T-cell,and the additional agent or combination thereof, comprised in a singlecomposition.

In one embodiment, methods disclosed herein comprise administering acomposition comprising a genetically modified cell, and the additionalagent or combination thereof, comprised in a at least two compositions.In another embodiment, methods comprise administering a compositioncomprising a CAR T-cell, and the additional agent or combinationthereof, comprised in at least two compositions. In another embodiment,methods comprise administering a composition comprising a TCR T-cell,and the additional agent or combination thereof, comprised in at leasttwo compositions.

For adoptive immunotherapy using antigen-specific T-cells, for exampleCAR T-cells, cell doses in the range of 10⁶-10¹⁰ (e.g., 10⁹) aretypically infused. Upon administration of the genetically modified cellsinto the host and subsequent differentiation, T-cells are induced thatare specifically directed against the specific antigen. “Induction” ofT-cells may include inactivation of antigen-specific T-cells such as bydeletion or anergy. Inactivation is particularly useful to establish orreestablish tolerance such as in autoimmune disorders. The modifiedcells can be administered by any method known in the art including, butnot limited to, intravenous, subcutaneous, intranodal, intratumoral,intrathecal, intrapleural, intraperitoneal and directly to the thymus.In some embodiments, the T-cells are not administered intraperitoneally.In some embodiments, the T-cells are administered intratumorallly.

Compositions comprising genetically modified immunoresponsive cells asdisclosed herein (e.g., T-cells, N cells, CTL cells, or theirprogenitors) can be provided systemically or directly to a subject forthe treatment of a neoplasia, pathogen infection, or infectious disease.In some embodiments, cells disclosed herein are directly injected intoan organ of interest (e.g., an organ affected by a neoplasia).Alternatively, compositions comprising genetically modifiedimmunoresponsive cells are provided indirectly to the organ of interest,for example, by administration into the circulatory system (e.g., thetumor vasculature). Expansion and differentiation agents can be providedprior to, during or after administration of the cells to increaseproduction of T-cells, NK cells, or CTL cells in vitro or in vivo.

As described above in methods disclosed herein, compositions comprisingadditional agents may be provided prior to, concurrent with, orfollowing administrations of the genetically modified immune cells. Inone embodiment, in methods disclosed herein genetically modified immunecells for example CAR T-cells are administered prior to an additionalagent as disclosed herein. In another embodiment, in methods disclosedherein genetically modified immune cells for example CAR T-cells areadministered concurrent with an additional agent, as disclosed herein.In another embodiment, in methods disclosed herein genetically modifiedimmune cells for example CAR T-cells are administered followingadministration of an additional agent.

In one embodiment, methods disclosed herein administer compositionscomprising apoptotic cells as disclosed herein. In another embodiment,methods disclosed herein administer compositions comprising apoptoticcell supernatants as disclosed herein.

The modified cells can be administered in any physiologically acceptablevehicle, normally intravascularly, although they may also be introducedinto bone or other convenient site where the cells may find anappropriate site for regeneration and differentiation (e.g., thymus).Usually, at least 1×10⁵ cells will be administered, eventually reaching1×10¹⁰ or more. Genetically modified immunoresponsive cells disclosedherein may comprise a purified population of cells. Those skilled in theart can readily determine the percentage of genetically modifiedimmunoresponsive cells in a population using various well-known methods,such as fluorescence activated cell sorting (FACS). In some embodiments,ranges of purity in populations comprising genetically modifiedimmunoresponsive cells are about 50 to about 55%, about 55 to about 60%,and about 65 to about 70%. In other embodiments, the purity is about 70to about 75%, about 75 to about 80%, about 80 to about 85%. In furtherembodiments, the purity is about 85 to about 90%, about 90 to about 95%,and about 95 to about 100%. Dosages can be readily adjusted by thoseskilled in the art (e.g., a decrease in purity may require an increasein dosage). The cells can be introduced by injection, catheter, or thelike. If desired, factors can also be included, including, but notlimited to, interleukins, e.g. IL-2, IL-3, IL-6, IL-11, IL7, IL12, ILIS,IL21, as well as the other interleukins, the colony stimulating factors,such as G-, M- and GM-CSF, interferons, e.g. gamma-interferon anderythropoietin.

Compositions include pharmaceutical compositions comprising geneticallymodified immunoresponsive cells or their progenitors and apharmaceutically acceptable carrier. Administration can be autologous orheterologous. For example, immunoresponsive cells, or progenitors can beobtained from one subject, and administered to the same subject or adifferent, compatible subject. Peripheral blood derived immunoresponsivecells disclosed herein or their progeny (e.g., in vivo, ex vivo or invitro derived) can be administered via localized injection, includingcatheter administration, systemic injection, localized injection,intravenous injection, or parenteral administration. When administeringa therapeutic composition as disclosed herein (e.g., a pharmaceuticalcomposition containing a genetically modified immunoresponsive cell), itwill generally be formulated in a unit dosage injectable form (solution,suspension, emulsion).

In another embodiment, disclosed herein is a method of producing acomposition comprising CAR T-cells or other immune cells as disclosedherein and apoptotic cells or an apoptotic cell supernatant, the methodcomprising introducing into the T-cell or immune cell the nucleic acidsequence encoding the CAR that binds to an antigen of interest. In analternative embodiment, the compositions comprising CAR T-cells or otherimmune cells as disclosed herein are separate from the compositioncomprising apoptotic cells or an apoptotic supernatant.

In one embodiment, disclosed herein is a method of treating, preventing,inhibiting, reducing the incidence of, ameliorating, or alleviating amalignancy comprising the step of administering a composition comprisingchimeric antigen receptor-expressing T-cells (CAR T-cells) and apoptoticcells or an apoptotic cell supernatant.

A skilled artisan would appreciate that an anti-tumor immunity responseelicited by the genetically modified immune cells, for exampleCAR-modified T cells, may be an active or a passive immune response. Inaddition, the CAR mediated immune response may be part of an adoptiveimmunotherapy approach in which CAR-modified T-cells induce an immuneresponse specific to the antigen binding moiety in the CAR.

A skilled artisan would appreciate that immunotherapeutics may encompassthe use of immune effector cells and molecules to target and destroycancer cells. The immune effector may be, for example, an antibodyspecific for some marker on the surface of a tumor cell. The antibodyalone may serve as an effector of therapy or it may recruit other cellsto actually effect cell killing. The antibody also may be conjugated toa drug or toxin (chemotherapeutic, radionuclide, ricin A chain, choleratoxin, pertussis toxin, etc.) and serve merely as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a tumorcell target. Various effector cells include cytotoxic T cells and NKcells.

In some embodiments, the early apoptotic cells and compositions thereof,as disclosed herein may be used to treat, prevent, inhibit the growthof, or reduce the incidence of, any hematological tumor known in theart. In some embodiments, the early apoptotic cells and compositionsthereof, as disclosed herein may be used to treat, prevent, inhibit thegrowth of, or reduce the incidence of, any diffuse cancer known in theart, for example but not limited to diffuse breast cancer, wherein asolid tumor is not formed in the breast. In some embodiments, the earlyapoptotic cells and compositions thereof, as disclosed herein may beused to extend the survival time of any hematological tumor known in theart. In some embodiments, the early apoptotic cells and compositionsthereof, as disclosed herein may be used to extend the survival time ofany diffuse cancer known in the art, for example but not limited todiffuse breast cancer, wherein a solid tumor is not formed in thebreast.

In some embodiments, the early apoptotic cells and compositions thereof,as disclosed herein may be used to increase the survival of a subjectsuffering from any hematological tumor known in the art. In someembodiments, the early apoptotic cells and compositions thereof, asdisclosed herein may be used to increase the survival of a subjectsuffering from any diffuse cancer known in the art, for example but notlimited to diffuse breast cancer, wherein a solid tumor is not formed inthe breast.

In some embodiments, the early apoptotic cells and compositions thereof,as disclosed herein may be used to reduce the growth rate of anyhematological tumor known in the art. In some embodiments, the earlyapoptotic cells and compositions thereof, as disclosed herein may beused to reduce the growth rate any diffuse cancer known in the art, forexample but not limited to diffuse breast cancer, wherein a solid tumoris not formed in the breast.

In some embodiments, the tumor or cancer being treated comprises ametastasis of a tumor or cancer. In some embodiments, methods of useherein prevent or reduce metastasis of a tumor or cancer. In someembodiments, methods of use herein inhibit the growth or reduce theincidence of metastasis.

In some embodiments, the subject is a human subject. In someembodiments, the subject is a child. In some embodiments, the subject isan adult. In some embodiments, the subject is animal mammal.

In some embodiments, a method disclosed herein comprises administeringan early apoptotic cell population comprising a mononuclear enrichedcell population, as described in detail above. In some embodiments, amethod disclosed herein comprises administering an early apoptotic cellpopulation comprising a stable population cell, wherein said cellpopulation is stable for greater than 24 hours. Stable populations ofearly apoptotic cells have been described in detail above. In someembodiments, a method disclosed herein comprises administering an earlyapoptotic cell population comprising a population of cells devoid ofcell aggregates. Early apoptotic cell populations devoid of aggregatesand methods of making them have been described in detail above.

In some embodiments, a method disclosed herein comprises administeringan autologous early apoptotic cell population to a subject in need. Insome embodiments, a method disclosed herein comprises administering anallogeneic early apoptotic cell population to a subject in need.

In some embodiments, methods of administration of early apoptotic cellpopulations or compositions thereof comprise administering a singleinfusion of said apoptotic cell population or composition thereof. Insome embodiments, a single infusion may be administered as aprophylactic to a subject predetermined to be at risk for a cancer ortumor. In some embodiments, a single infusion may be administered to asubject having a cancer or tumor on a regular basis as a part of thesubject therapeutic treatment. In some embodiments, a single infusionmay be administered as a prophylactic to a subject having a cancer ortumor in order to prevent, reduce the risk of, or delay the appearanceof metastatic cancer.

In some embodiments, methods of administration of early apoptotic cellpopulations or compositions thereof comprise administering multipleinfusions of said apoptotic cell population or composition thereof. Insome embodiments, multiple infusions may be administered as aprophylactic to a subject predetermined to be at risk for a cancer ortumor. In some embodiments, multiple infusions may be administered to asubject having a cancer or tumor on a regular basis as a part of thesubject therapeutic treatment. In some embodiments, multiple infusionsmay be administered as a prophylactic to a subject having a cancer ortumor in order to prevent, reduce the risk of, or delay the appearanceof metastatic cancer.

In some embodiments, multiple infusions comprise at least two infusions.In some embodiments, multiple infusions comprise 2 infusions. In someembodiments, multiple infusions comprise more than 2 infusions. In someembodiments, multiple infusions comprise at least 3 infusions. In someembodiments, multiple infusions comprise 3 infusions. In someembodiments, multiple infusions comprise more than 3 infusions. In someembodiments, multiple infusions comprise at least 4 infusions. In someembodiments, multiple infusions comprise 4 infusions. In someembodiments, multiple infusions comprise more than 4 infusions. In someembodiments, multiple infusions comprise at least 5 infusions. In someembodiments, multiple infusions comprise 5 infusions. In someembodiments, multiple infusions comprise more than 5 infusions. In someembodiments, multiple infusions comprise at least six infusions. In someembodiments, multiple infusions comprise 6 infusions. In someembodiments, multiple infusions comprise more than 6 infusions. In someembodiments, multiple infusions comprise at least 7 infusions. In someembodiments, multiple infusions comprise 7 infusions. In someembodiments, multiple infusions comprise more than 7 infusions. In someembodiments, multiple infusions comprise at least 8 infusions. In someembodiments, multiple infusions comprise 8 infusions. In someembodiments, multiple infusions comprise more than 8 infusions. In someembodiments, multiple infusions comprise at least nine infusions. Insome embodiments, multiple infusions comprise 9 infusions. In someembodiments, multiple infusions comprise more than 9 infusions. In someembodiments, multiple infusions comprise at least 10 infusions. In someembodiments, multiple infusions comprise 10 infusions. In someembodiments, multiple infusions comprise more than 10 infusions.

In some embodiments, multiple infusions comprise smaller amounts ofearly apoptotic cell, wherein the total dosage of cells administered isthe sum of the infusions.

In some embodiments, multiple infusions are administered over a periodof hours. In some embodiments, multiple infusions are administered overa period of days. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least 12 hoursbetween infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least 24 hoursbetween infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least a daybetween infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least two daysbetween infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least threedays between infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least four daysbetween infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least five daysbetween infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least six daysbetween infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least sevendays between infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least a weekbetween infusions. In some embodiments, multiple infusions areadministered over a period of hours, wherein there is at least two weeksbetween infusions.

In some embodiments, the amount of cells in multiple infusions isessentially equivalent one to the other. In some embodiments, the amountof cells in multiple infusions is different one to the other.

In some embodiments, the methods described herein further compriseadministering an additional chemotherapeutic agent or an immunemodulator to said subject.

In some embodiments, an additional chemotherapeutic agent or immunemodulator is administered concurrent or essentially concurrent with theearly apoptotic cells. In some embodiments, an additionalchemotherapeutic agent or immune modulator is comprised in the samecomposition as the early apoptotic cells. In some embodiments, anadditional chemotherapeutic agent or immune modulator is comprised in adifferent composition as the early apoptotic cells.

In some embodiments, an additional chemotherapeutic agent or immunemodulator is administered prior to the administration of the earlyapoptotic cells. In some embodiments, an additional chemotherapeuticagent or immune modulator is administered following the administrationof the early apoptotic cells.

In some embodiments, the chemotherapeutic agent comprises alkylatingagents, nitrogen mustards, nitrosoureas, tetrazines, aziridines,cisplatins and derivatives, non-classical alkylating agents,mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide,busulfan, N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine(CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine,mitozolomide, temozolomide, thiotepa, mitomycin, diaziquone (AZQ),cisplatin, carboplatin, oxaliplatin, procarbazine, hexamethylmelamine,antimetabolites, anti-folates, methotrexate, pemetrexed,fluoropyrimidines, fluorouracil, capecitabine, deoxynucleosideanalogues, cytarabine, gemcitabine, decitabine, azacitidine,fludarabine, nelarabine, cladribine, clofarabine, and pentostatin,thiopurines, thioguanine, mercaptopurine, anti-microtubule agents, vincaalkaloids, taxanes, vincristine, vinblastine, semi-synthetic vincaalkaloids, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel,podophyllotoxin, etoposide, teniposide, topoisomerase inhibitors,irinotecan, topotecan, camptothecin, etoposide, doxorubicin,mitoxantrone, teniposide, catalytic inhibitors, novobiocin, merbarone,aclarubicin, cytotoxic antibiotics, anthracyclines, bleomycins,mitomycin C, mitoxantrone, actinomycin, doxorubicin, daunorubicin,epirubicin, idarubicin, anthracyclines, pirarubicin, aclarubicin,mitoxantrone, bleomycin, mitomycin, targeted therapies, monoclonalantibodies, naked monoclonal antibodies, conjugated monoclonalantibodies, chemolabeled antibodies, bispecific monoclonal antibodies,or any combination thereof.

In some embodiments, an immune modulator comprises an antibody or afunctional fragment thereof. In some embodiments, an antibody orfunctional fragment thereof comprises a monoclonal antibody, a singlechain antibody, an Fab fragment, an F(ab′)2 fragment, or an Fv fragment.

In some embodiments, disclosed herein are active fragments of any one ofthe polypeptides or peptide domains disclosed herein. A skilled artisanwould appreciate that the term “a fragment” may encompass at least 5,10, 13, or 15 amino acids. In other embodiments a fragment is at least20 contiguous amino acids. Fragments disclosed herein can be generatedby methods known to those skilled in the art or may result from normalprotein processing (e.g., removal of amino acids from the nascentpolypeptide that are not required for biological activity or removal ofamino acids by alternative mRNA splicing or alternative proteinprocessing events).

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and specifically refer to a polyclonal antibody, amonoclonal antibody, or any fragment thereof, which retains the bindingactivity of the antibody. In certain embodiments, methods disclosedherein comprise use of a chimeric antibody, a humanized antibody, or ahuman antibody.

In some embodiments, the term “antibody” refers to intact molecules aswell as functional fragments thereof, such as Fab, F(ab′)2, and Fv thatare capable of specifcially interacting with a desired target asdescribed herein, for example, binding to phagocytic cells. In someembodiments, the antibody fragments comprise:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, which can be produced by digestion ofwhole antibody with the enzyme papain to yield an intact light chain anda portion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule that can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)2, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)2 is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference).

In some embodiments, the antibody fragments may be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment.

Antibody fragments can, in some embodiments, be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)2. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein, which patents are herebyincorporated by reference in their entirety. See also Porter, R. R.,Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies,such as separation of heavy chains to form monovalent light-heavy chainfragments, further cleavage of fragments, or other enzymatic, chemical,or genetic techniques may also be used, so long as the fragments bind tothe antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, which is hereby incorporated by reference inits entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry,Methods, 2: 106-10, 1991.

In some embodiments, the antibodies or fragments as described herein maycomprise “humanized forms” of antibodies. In some embodiments, the term“humanized forms of antibodies” refers to non-human (e.g. murine)antibodies, which are chimeric molecules of immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′). sub.2 or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues form a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., Mol. Biol., 222:581 (1991)].The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol., 147(1):86-95 (1991)]. Similarly, human can be made byintroducing of human immunoglobulin loci into transgenic animals, e.g.mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. Upon challenge, human antibody production isobserved, which closely resembles that seen in humans in all respects,including gene rearrangement, assembly, and antibody repertoire. Thisapproach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in thefollowing scientific publications: Marks et al., Bio/Technology 10,779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison,Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14,845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonbergand Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

In some embodiments, the immune modulator comprises an anti-CD20monoclonal antibody. In some embodiments, the antiCD20 monoclonalantibody is Rituximab. Rituximab is commercially available and soldunder the name Rituxan®, marketed jointly by Biogen and Genentech USA,Inc.

In some embodiments, methods disclosed herein comprise a first-linetherapy.

A skilled artisan would appreciate that the term “first-line therapy”may encompass the first treatment given for a disease. It is often partof a standard set of treatments, such as surgery followed bychemotherapy and radiation. When used by itself, first-line therapy isthe one accepted as the best treatment. If it doesn't cure the diseaseor it causes severe side effects, other treatment may be added or usedinstead. Also called induction therapy, primary therapy, and primarytreatment.

In some embodiments, methods disclosed herein comprise an adjuvanttherapy.

A skilled artisan would appreciate that the term “adjuvant therapy” mayencompass a treatment that is given in addition to the primary orinitial treatment. In some embodiments, adjuvant therapy may comprise anadditional cancer treatment given prior to the primary treatment inpreparation of a further treatment. In some embodiments, adjuvanttherapy may comprise an additional cancer treatment given after theprimary treatment to lower the risk that the cancer will come back.Adjuvant therapy may include chemotherapy, radiation therapy, hormonetherapy, targeted therapy, or biological therapy.

In some embodiments, a method disclosed herein, reduces the minimalresidual disease, increases remission, increases remission duration,reduces tumor relapse rate, decreases the size of said tumor, decreasesgrowth rate of said tumor or said cancer, prevents metastasis of saidtumor or said cancer, or reduces the rate of metastasis of said tumor orsaid cancer, or any combination thereof.

A skilled artisan would appreciate that the term “minimal residualdisease” may encompass small numbers of cancer cells that remain in thepatient during treatment or after treatment when the patient has nosymptoms or signs of disease.

Additionally, the term “remission” may encompass a decrease ordisappearance of signs and symptoms of cancer, though cancer may stillbe in the body. In some embodiments, remission may comprise partialremission, wherein some, but not all, signs and symptoms of cancer havedisappeared. In some embodiments, remission comprises completeremission, wherein all signs and symptoms of cancer have disappeared,although cancer still may be in the body. In some embodiments, methodsdisclosed herein may be comprise a remission induction therapy, whereinthe initial treatment with early apoptotic cells or compositionsthereof, decreases the signs or symptoms of cancer or make themdisappear.

A skilled artisan would appreciate that the term “relapse” may encompassthe return of a disease or the signs and symptoms of a disease after aperiod of improvement. In some embodiments, methods used herein lead toa relapse-free survival, wherein the relapse-free survival encompassesthe length of time after primary treatment for a cancer ends that thepatient survives without any signs or symptoms of that cancer.

A skilled artisan would appreciate that the term “metastasis”encompasses the spread of cancer cells from the place where they firstformed to another part of the body. In metastasis, cancer cells breakaway from the original (primary) tumor, travel through the blood orlymph system, and form a new tumor in other organs or tissues of thebody. In some embodiments, the new, metastatic tumor is the same type ofcancer as the primary tumor. For example, if breast cancer spreads tothe lung, the cancer cells in the lung are breast cancer cells, not lungcancer cells.

Malignancies

In some embodiments, CAR T-cells are utilized in methods of treating,preventing, inhibiting, reducing the incidence of, ameliorating, oralleviating a cancer or a tumor wherein the methods comprise the step ofadministering chimeric antigen receptor-expressing T-cells (CART-cells). As disclosed herein, these methods may further compriseadministering an additional agent in an effort to inhibit or decreasethe incidence of CRS or cytokine storm.

In some embodiments, a method disclosed herein increases the survival ofthe subject. In some embodiments, disclosed herein is a method ofincreasing or lengthening the survival of a subject having a diffusecancer, comprising the step of administering an early apoptotic cellpopulation to said subject, wherein the method increases the survival ofthe subject.

In some embodiments, a cancer is a B-cell malignancy. In someembodiments, the B-cell malignancy is leukemia. In another embodiment,the B-cell malignancy is acute lymphoblastic leukemia (ALL). In anotherembodiment, the B-cell malignancy is chronic lymphocytic leukemia.

In some embodiments, the cancer is leukemia. In some embodiments, thecancer is lymphoma. In some embodiments, the lymphoma is large B-celllymphoma.

In some embodiments, methods described herein reduce the size or reducethe growth rate of a cancer or a tumor, and comprise administering anearly apoptotic cell population to said subject, wherein the methodreduces the size or the growth rate of a cancer or tumor. In someembodiments, disclosed herein is a method of reducing the growth rate ofa diffuse cancer, comprising the step of administering an earlyapoptotic cell population to said subject, wherein the method reducesthe growth rate of the cancer. In some embodiments, disclosed herein isa method of reducing the size or reducing the growth rate of a solidcancer or tumor, comprising the step of administering an early apoptoticcell population to a subject, wherein the method reduces the size orreduces the growth rate of the solid cancer or tumor.

In some embodiments, a cancer may comprise a solid tumor. In someembodiments, a solid tumor comprises an abnormal mass of tissue thatusually does not contain cysts or liquid areas. Solid tumors may bebenign (not cancer), or malignant (cancer). Different types of solidtumors are named for the type of cells that form them. Examples of solidtumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers ofthe blood) generally do not form solid tumors. In some embodiments, asolid tumor comprises a sarcoma or a carcinoma.

In some embodiments, solid tumors are neoplasms (new growth of cells) orlesions (damage of anatomic structures or disturbance of physiologicalfunctions) formed by an abnormal growth of body tissue cells other thanblood, bone marrow or lymphatic cells. In some embodiments, a solidtumor consists of an abnormal mass of cells which may stem fromdifferent tissue types such as liver, colon, breast, or lung, and whichinitially grows in the organ of its cellular origin. However, suchcancers may spread to other organs through metastatic tumor growth inadvanced stages of the disease.

In some embodiments, examples of solid tumors comprise sarcomas,carcinomas, and lymphomas. In some embodiments, a solid tumor comprisesa sarcoma or a carcinoma. In some embodiments, the solid tumor is anintra-peritoneal tumor.

In some embodiments, a solid tumor comprises, but is not limited to,lung cancer, breast cancer, ovarian cancer, stomach cancer, esophagealcancer, cervical cancer, head and neck cancer, bladder cancer, livercancer, and skin cancer. In some embodiments, a solid tumor comprises afibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, anosteogenic sarcoma, a chordoma, an angiosarcoma, an endotheliosarcoma, alymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, amesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, acolon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor,an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cellcarcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat glandcarcinoma, a sebaceous gland carcinoma, a papillary carcinoma, apapillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma,a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bileduct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, aWilm's tumor, a cervical cancer or tumor, a uterine cancer or tumor, atesticular cancer or tumor, a lung carcinoma, a small cell lungcarcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, anastrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, apinealoma, a hemangioblastoma, an acoustic neuroma, an oligodenroglioma,a schwannoma, a meningioma, a melanoma, a neuroblastoma, or aretinoblastoma.

In some embodiments, the solid tumor comprises an Adrenocortical Tumor(Adenoma and Carcinoma), a Carcinoma, a Colorectal Carcinoma, a DesmoidTumor, a Desmoplastic Small Round Cell Tumor, an Endocrine Tumor, anEwing Sarcoma, a Germ Cell Tumor, a Hepatoblastoma a HepatocellularCarcinoma, a Melanoma, a Neuroblastoma, an Osteosarcoma, aRetinoblastoma, a Rhabdomyosarcoma, a Soft Tissue Sarcoma Other ThanRhabdomyosarcoma, and a Wilms Tumor. In some embodiments, the solidtumor is a breast tumor. In another embodiment, the solid tumor is aprostate cancer. In another embodiment, the solid tumor is a coloncancer. In some embodiments, the tumor is a brain tumor. In anotherembodiment, the tumor is a pancreatic tumor. In another embodiment, thetumor is a colorectal tumor.

In some embodiments, early apoptotic cells or compositions thereof asdisclosed herein, have therapeutic and/or prophylactic efficacy againsta cancer or a tumor, for example sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).

In some embodiments, the early apoptotic cells and compositions thereofas disclosed herein may be used to treat, prevent, inhibit the growthof, or reduce the incidence of, any solid tumor known in the art.

In some embodiments, the early apoptotic cells and compositions thereofas disclosed herein, may be used to increase the survival of a subjectsuffering from any solid tumor as disclosed herein or known in the art.

In some embodiments, the early apoptotic cells and compositions thereofas disclosed herein, may be used to reduce the size or reduce the growthrate any solid tumor as disclosed herein or known in the art.

In some embodiments, a cancer may be a diffuse cancer, wherein thecancer is widely spread; not localized or confined. In some embodiments,a diffuse cancer may comprise a non-solid tumor. Examples of diffusecancers include leukemias. Leukemias comprise a cancer that starts inblood-forming tissue, such as the bone marrow, and causes large numbersof abnormal blood cells to be produced and enter the bloodstream.

In some embodiments, a diffuse cancer comprises a B-cell malignancy. Insome embodiments, the diffuse cancer comprises leukemia. In someembodiments, the cancer is lymphoma. In some embodiments, the lymphomais large B-cell lymphoma.

In some embodiments, the diffuse cancer or tumor comprises ahematological tumor. In some embodiments, hematological tumors arecancer types affecting blood, bone marrow, and lymph nodes.Hematological tumors may derive from either of the two major blood celllineages: myeloid and lymphoid cell lines. The myeloid cell linenormally produces granulocytes, erythrocytes, thrombocytes, macrophages,and masT-cells, whereas the lymphoid cell line produces B, T, NK andplasma cells. Lymphomas (e.g. Hodgkin's Lymphoma), lymphocyticleukemias, and myeloma are derived from the lymphoid line, while acuteand chronic myelogenous leukemia (AML, CML), myelodysplastic syndromesand myeloproliferative diseases are myeloid in origin.

In some embodiments, a non-solid (diffuse) cancer or tumor comprises ahematopoietic malignancy, a blood cell cancer, a leukemia, amyelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cellmyeloma), an acute lymphoblastic leukemia, an acute myelogenousleukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, anon-Hodgkin lymphoma, or plasma cell leukemia.

In another embodiment, early apoptotic cells and compositions thereof,as disclosed herein have therapeutic and/or prophylactic efficacyagainst diffuse cancers, for example but not limited to leukemias (e.g.,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia,acute myeloblastic leukemia, acute promyelocyte leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease.

The compositions and methods as disclosed herein may be used to treat,prevent, inhibit, ameliorate, reduce the incidence of, or alleviate anyhematological tumor known in the art.

A skilled artisan would appreciate that the use of the term comprisingthroughout, may in certain embodiments be replace by the use of the termconsisting essentially of or consisting of. The skilled artisan wouldappreciate that the term “comprising” is intended to mean that thesystem includes the recited elements, but not excluding others which maybe optional. For example a composition comprising early apoptotic cellsbut not limited to this population of cells. Further, the term“consisting essentially of” may encompass a method that includes therecited elements, for example a composition consisting essentially orearly apoptotic cells but exclude other elements that may have anessential significant effect on the performance of the method. Thus,such a composition may still include a pharmaceutically acceptableexcipient that does not comprise an essential activity in treatingcancer. Further, “consisting of” encompasses excluding more than tracesof other elements. Thus, such a composition consisting of earlyapoptotic cells would not include more than traces of other elements asdisclosed herein.

In some embodiments, methods as disclosed herein may be represented asuses of the compositions as described herein for various therapeutic andprophylactic purposes as described herein, or alternatively, uses of thecompositions as described herein in the preparation of a medicament or atherapeutic composition or a composition for various therapeutic andprophylactic purposes as described herein.

In some embodiments, the compositions and methods as disclosed hereincomprise the various components or steps. However, in anotherembodiment, the compositions and methods as disclosed herein consistessentially of the various components or steps, where other componentsor steps may be included. In another embodiment, the compositions andmethods as disclosed herein consist of the various components or steps.

In some embodiments, the term “comprise” may encompass the inclusion ofother components of the composition which affect the efficacy of thecomposition that may be known in the art. In some embodiments, the term“consisting essentially of” comprises a composition, which has chimericantigen receptor-expressing T-cells (CAR T-cells), and apoptotic cellsor any apoptotic cell supernatant. However, other components may beincluded that are not involved directly in the utility of thecomposition. In some embodiments, the term “consisting” encompasses acomposition having chimeric antigen receptor-expressing T-cells (CART-cells), and apoptotic cells or an apoptotic cell supernatant asdisclosed herein, in any form or embodiment as described herein.

A skilled artisan would appreciate that the term “about”, may encompassa deviance of between 0.0001-5% from the indicated number or range ofnumbers. Further, it may encompass a deviance of between 1-10% from theindicated number or range of numbers. In addition, it may encompass adeviance of up to 25% from the indicated number or range of numbers.

A skilled artisan would appreciate that the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “an agent” or “at least an agent” mayinclude a plurality of agents, including mixtures thereof.

Throughout this application, various embodiments disclosed herein may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicated number and asecond indicated number and “ranging/ranges from” a first indicatednumber “to” a second indicated number are used herein interchangeablyand are meant to include the first and second indicated numbers and allthe fractional and integral numerals there between.

The following examples are presented in order to more fully illustrateembodiments disclosed herein. They should in no way be construed,however, as limiting the broad scope of the disclosure.

EXAMPLES Example 1: Apoptotic Cell Production

Objective:

To produce early-apoptotic cells.

Methods:

Methods of making populations of early-apoptotic cells have been welldocumented in International Publication No. WO 2014/087408 and UnitedStates Application Publication No. US2015/0275175-A1, see for example,the Methods section preceding the Examples at “Early apoptotic cellpopulation Preparation” and “Generation of apoptotic cells” (paragraphs[0223] through [0288]), and Examples 11, 12, 13, and 14, which areincorporated herein in their entirety).

The flow chart presented in FIG. 1 provides an overview of oneembodiment of the steps used during the process of producing apopulation of early apoptotic cells, wherein anticoagulants wereincluded in the thawing and induction of apoptosis steps. As isdescribed in detailed in Example 14 of International Publication No. WO2014/087408 and United States Application Publication No. USUS-2015-0275175-A1, early apoptotic cell populations were preparedwherein anti-coagulants were added at the time of freezing, or at thetime of incubation, or at the time of freezing and at the time ofincubation. The anticoagulant used was acid-citrate dextrose, NIHFormula A (ACD formula A) was supplemented with 10 U/ml heparin to afinal concentration of 5% ACD of the total volume and 0.5 U/ml heparin.

Briefly: The cells were collected and then frozen with addition of 5%anticoagulant citrate dextrose formula A and 10 U/ml heparin (ACDhep) tothe freezing media. Thawing, incubation in an apoptosis induction mediacontaining 5% ACDhep, and final product preparation were performed in aclosed system.

Apoptosis and viability analysis, potency assay, and cell populationcharacterization were performed in each experiment. In order toestablish consistence in production of the early apoptotic cell product,the final product (FP) of initial batches of apoptotic cells were storedat 2-8° C. and examined at t0, t24 h, t48 h and t72 h. At each pointapoptosis analysis, short potency assay (Applicants CD14+ frozen cells),trypan blue measurement and cell population characterization wereperformed. The FP was tested for cell count to assess average cell lossduring storage and apoptosis and viability analysis.

The methods sections cited above and Example 11 of InternationalPublication No. WO 2014/087408 and United States Application PublicationNo. US US-2015-0275175-A1 provide details of preparing other embodimentof apoptotic cell populations in the absence of anti-coagulants, and areincorporated herein in full.

Methods of Preparing Irradiated Apoptotic Cells:

Similar methods were used to prepare an inactivated apoptotic cellpopulation, wherein a mononuclear early apoptotic cell populationcomprises a decreased percent of non-quiesnce non-apoptoic cells, or apopulation of cells having a suppressed cellular activation of anyliving non-apoptotic cells, or a population of cells having a reducedproliferation of any living non-apoptotic cells, or any combinationthereof.

Briefly, an enriched mononuclear cell fraction was collected vialeukapheresis procedure from healthy, eligible donors. Followingapheresis completion, cells were washed and resuspended with freezingmedia comprising 5% Anticoagulant Citrate Dextrose Solution-Formula A(ACD-A) and 0.5 U\ml heparin. Cell were then gradually frozen andtransferred to liquid nitrogen for long term storage.

For preparation of irradiated ApoCells, cryopreserved cells were thawed,washed and resuspended with apoptosis induction media comprising 5%ACD-A, 0.5 U\ml heparin sodium and 50 μg/ml methylprednisolone. Cellswere then incubated for 6 hours at 37° C. in 5% CO₂. At the end ofincubation, cells were collected, washed and resuspended in Hartmann'ssolution using a cell processing system (Fresenius Kabi, Germany).Following manufacturing completion, ApoCell were irradiated at 4000 cGyusing g-camera at the radiotherapy unit, Hadassah Ein Kerem. Apoptosisand viability of ApoCell determined using AnnexinV and PI (MBL, MA, USA)staining (≥40% and ≤15%, respectively) via Flow cytometer. Resultsanalyzed using FCS express software. This irradiated APOcell populationis considered to include early apoptotic cells, wherein any viable cellspresent have suppressed cellular activity and reduced or noproliferation capabilities. In certain cases, the Apocell population hasno viable non-apoptotic cells. Results:

The stability of the FP produced with inclusion of anticoagulant atfreezing and incubation (apoptotic induction) and then stored at 2-8° C.are shown below in Table 3.

TABLE 3 Cell count*—performed using a MICROS 60 hematology analyzer.Cell concentration FP Time point (× 10⁶ cells\ml) % of cell loss t0 20.8NA t24 h 20.0 −3.85 t48 h 20.0 −3.85 t72 h 19.7 −5.3  *ResultsRepresentative of 6 (six) experiments.

When manufacturing the cells without including an anticoagulant in theinduction medium, cells were stable for 24 hours and less stablethereafter. Use of anticoagulants unexpectedly extended the stability ofthe apoptotic cell population for at least 72 hours, as shown in Table3.

TABLE 4 Trypan blue measurement FP Time point trypan blue positive cells(%) t0 3.0 t24 h 5.9 t48 h 5.2 t72 h 6.5

The results of Table 4 show viability of the FP remained high for atleast 72 hours.

TABLE 5 Apoptosis analysis—(AnPI staining) performed using FlowCytometry FP Time 1.5 mM Ca point An−PI− (%) An+PI− (%) An+PI+ (%) t044.3 50.9 4.8 t24 h 39.0 55.9 5.1 t48 h 34.8 60.1 5.1 t72 h 33.4 60.56.1

The results of Table 5 show that the percent apoptotic cells versusnecrotic cells was maintained over at extended time period of at least72 hours post preparation of the cells, as was the percentage of earlyapoptotic cells.

Inclusion of anticoagulants both at the time of freezing and duringinduction of apoptosis resulted in the most consistently high yield ofstable early-apoptotic cells (average yield of early apoptotic cells61.3±2.6% % versus 48.4±5.0%, wherein 100% yield is based on the numberof cells at freezing). This high yield was maintained even after 24hours storage at 2-8° C.

Next a comparison was made between the inclusion of the anticoagulant atfreezing or thawing or both, wherein percent (%) recovery was measuredas well as stability. Anticoagulant was included in the apoptoticincubation mix for all populations. Table 6 presents the results ofthese studies.

TABLE 6 Yield and stability comparison of final products (FP)manufactured from cells collected, with (“+”) or without (“−”) additionof anticoagulant during freezing (“F”) and thawing (“Tha”) # ofCollected % Cell Recovery in Final Product of Collected Cells DonorCells FP t0 FP t24 h* ID (×10⁹, 100%) F−/Tha− F−/Tha+ F+/Tha+ F+/Tha−F−/Tha− F−/Tha+ F+/Tha+ F+/Tha− 1 13.3 52.1 53.4 62.5 62 52.1 48.9 62.562 2 13.6 50.5 36.7 53.5 63.5 47.6 36.7 53.1 59.7 3 15.0 42.7 42 53.658.4 42.7 41.7 53.6 57.8 Avg 14.0 48.4 ± 5.0 44.0 ± 8.5 56.5 ± 5.2 61.3± 2.6 47.5 ± 4.7 42.4 ± 6.1 56.4 ± 5.3 59.8 ± 2.1

Additional population analysis comparisons of early apoptotic cellpopulations (batches of cells) prepared with and without anti-coagulantadded, show the consistency of these results.

TABLE 7 Cell population analysis comparison between batches preparedwith and without anticoagulant ApoCell ApoCell At Thawing Time 0 h Time24 h Storage w\o w\o w\o Test Specification ACDhep +ACDhep ACDhep+ACDhep ACDhep +ACDhep Change in Total  >35.0% 85.5  82.8 49.9 66.7 49.066.7 Cell Count (79.5-92.5) (67.7-96.4) (46.6-52.3) (62.5-71.2) 46.6-50.3) (62.5-71.2) Percent change (min-max) Changes in 90.0 ± 10.0%100   100   98.2 100   ApoCell (96.2-100)  Percent change Range(min-max) Cell viability PI  >85.0% 98.0  96.0 98.5 94.6 97.7 94.5exclusion (97.4-98.4) (91.9-98.1) (97.9-99.2) (93.5-95.5) (96.4-98.6)(93.4-95.1) Percent viable Range (min-max) Identity/ CD3 (T cells):75.7  66.5 73.3 62.8 71.6 64.2 Purity 71.9 (71.6-81.4) (60.1-70.1)(70.3-78.3) (61.1-65.3) (61.5-79.1) (61.6-68.1) Analysis of cell(50.0-85.0)  phenotype Average (%) ApoCell CD3: (maximal 71.6 calculated(50.0-85.0)  range) CD19 (B cells): 7.5  9.8  9.0  9.9  9.5  9.7  9.3 (4.0-11.1)  (8.6-12.0)  (7.6-10.2)  (9.3-10.2)  (8.6-10.3)  (9.2-10.4)(3.0-15.0) ApoCell CD19:  9.5 (4-15) CD14 9.8 14.0 11.6 15.4  9.3 16.1(monocytes):  (6.4-13.0)  (8.8-22.1) (10.2-13.3)  (8.2-19.3)  (4.8-17.2) (9.0-20.4) 10.1 (2.5-22.0) ApoCell CD14: 10.6 (2.5-22.0) CD15^(high)0.2  0.46  0.2   0.083  0.1  0.09 (granulocytes):   (0-0.3) (0.18-0.69)(0.1-0.4) (0.08-0.09) (0.1-0.2) (0.07-0.1)   0.4  (0-6.0) ApoCellCD15^(high):  0.2  (0-2.0) CD 56 (NK): 7.4 10.1  4.7 11.2  4.9 10.0  7.2 (2.4-11.0)  (6.6-14.2) (2.7-8.0)  (7.2-14.2) (2.2-9.2)  (6.4-13.0)(1.5-22.0) ApoCell CD56:  5.2 (1.5-15.0)

Percentage of final product cells (yield) in the presence or absence ofanticoagulants. Similar to the results presented above at Table 3, thedata presented in Table 6 demonstrates that early apoptotic cellsmanufactured from cells frozen in the presence of anticoagulant had abeneficial effect on average yield of fresh final product (FP t0) ascompared to cells frozen without anticoagulant. The beneficial effectwas seen when anticoagulant was used while freezing only (61.3±2.6%versus 48.4±5.0%), or both freezing and thawing (56.5±5.2% versus48.4±5.0%). The beneficial effect was less significant whenanticoagulant was used upon thawing only (44.0±8.5% versus 48.4±5.0%).These were non-high triglyceride samples.

Effect of Anticoagulants on Aggregation.

No cell aggregations were seen in these 3 non-high triglyceride samples,or in 21 additional samples (data not shown). However, in 41 othernon-high triglyceride samples manufactured without anticoagulants (datanot shown), mild aggregates were seen in 10 (24.4%) and severeaggregates in 5 (12.2%); thus, anticoagulants avoid completely cellaggregates.

Effect of Anticoagulants on Stability.

Fresh FPs manufactured with- or without anticoagulants were stored at2-8° C. for 24 hours to determine whether addition of ACDhep to themanufacturing procedure impairs the stability of the FP. Cells weresampled following 24 hours of storage and yield was calculated In cellcount. Similar to the results shown in Table 3 for extended time periods(up to 72 hours), Table 6 shows that the beneficial effect was kept andobserved when anticoagulant was used while freezing only (59.8±2.1%versus 47.5±4.7%), or both freezing and thawing (56.4±5.3% versus47.5±4.7%). The beneficial effect was less significant whenanticoagulant was added only upon thawing (42.4±6.1% versus 47.5±4.7%).These were all non-high triglyceride samples. These results show minimalcell loss following 24 hours of FP storage in all treatments withsignificant advantage to cells treated with anticoagulant during bothfreezing and thawing. Average loss of cells treated with anticoagulantduring freezing only was 2.3±3.2% compared to 1.9±3.3% withoutanticoagulants, upon thawing only was 3.0±4.7 compared to 1.9±3.3%without anticoagulants, and 0.2±0.4% compared to 1.9±3.3% withoutanticoagulants when cells were both frozen and thawed with ACDhep. Insummary, the beneficial effect of anticoagulants on yield was kept forat least 24 hours.

The characteristics of a representative cell population of the FP areshown below in Table 8.

TABLE 8 Characterization of the cell population of fresh (t0) FPmanufactured from cells collected with (“+”) or without (“−”) additionof anticoagulant during freezing (“F”) and thawing (“Tha”) procedures.*FP t0 F−/Tha− F−/Tha+ Donor CD3+ CD19+ CD56+ CD14+ CD15+ CD3+ CD19+CD56+ CD14+ CD15+ ID (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) 1-3 62.2 ±6.1 5.6 ± o.7 9.8 ± 0.9 13.5 ± 1.1 0 ± 0 61 ± 6.1 8.6 ± 0.4 8.6 ± 0.914.1 ± 1.1 0 ± 0 FP t0 F+/Tha+ F+/Tha− Donor CD3+ CD19+ CD56+ CD14+CD15+ CD3+ CD19+ CD56+ CD14+ CD15+ ID (%) (%) (%) (%) (%) (%) (%) (%)(%) (%) 1-3 63.9 ± 5.8 7.4 ± 0.6 9.4 ± 0.8 13.3 ± 1.9 0 ± 0 61.9 ± 6.011.5 ± 1.1 10.1 ± 1.0 14.3 ± 1.3 0 ± 0 *Induction of apoptosis wasperformed using a medium containing anticoagulant for all batches.

The results of Table 8 show the cell characteristics of the finalproducts (FP) manufactured with or without anticoagulant at freezing andthawing. Batches were sampled, stained for mononuclear markers, andanalyzed via flow cytometry to determine the cell distribution in eachsample and to examine whether the addition of anticoagulant affected thecell population. As presented in Table 7, there were no significantdifferences detected in cell populations manufactured with or withoutanticoagulants at freezing or thawing. The average T cell population(CD3+ cells) in fresh FP was 62.3±1.2% between treatments compared to62.9±1.1% before freezing; the average B cell population (CD19+ cells)was 8.3±2.5% between treatments compared to 3.1±40.8% before freezing;the average natural killer cell population (CD56+ cells) was 9.5±0.7%between treatments compared to 12.9±0.5% before freezing; the averagemonocyte cell population (CD14+ cells) was 13.8±0.5% between treatmentscompared to 17.5±0.3% before freezing; and the average granulocytepopulation (CD15+ cells) was 0.0% in the fresh FP compared to 0.35±0.2%at freezing.

The potency of the early apoptotic population was also examined.

TABLE 9 Potency analysis of fresh (t0) FP manufactured from cells with(“+”) or without (“−”) addition of anticoagulant during freezing (“F”)and thawing (“Tha”) procedures. FP t0 Treatment F−/Tha− F−/Tha+ F+/Tha+F+/Tha− Donor Median fluorescence ID # DR CD86 DR CD86 DR CD86 DR CD86DCs 1:2 Early 3% 28% 4% up 24% 5% 24% 9% 15% apoptotic cell frompopulation + LPS LPS DCs 1:4 Early 4% 38%  6% 35% 6% 34% 6% 24%apoptotic cell population + LPS DCs 1:8 Early 13%  Not 10% 45% 15%  54%8% 48% apoptotic cell done population + LPS

The results presented in Table 9 are from a potency assay performed todetermine the ability of each final product to enhance a tolerogenicstate in immature dendritic cells (iDCs) following stimulation with(LPS). The tolerogenic effect was determined by assessing downregulationof co-stimulatory molecule HLA-DR and CD86 expression on iDCs followinginteraction with the early apoptotic cell populations and differenttreatments leading to LPS upregulation. The analysis was performed onDCsign+ cells. Results represent the percent delay in maturationfollowing interaction with early apoptotic cell population and followingaddition of LPS versus LPS-induced maturation. The experiment tested thepotency of fresh FP (t0) manufactured with- or without anticoagulant.Results presented in Table 9 show that apoptotic cells manufactured withor without anticoagulant enhance the tolerance effect of bothco-stimulatory markers in a dose-dependent manner.

The early apoptotic cells produced herein were from non-hightriglyceride samples. This consistent high yield of stable earlyapoptotic cells was produced even in the cases when the donor plasma ishigh in triglycerides (See for example, Examples 12 and 13 ofInternational Publication No. WO 2014/087408 and United StatesApplication Publication No. US US-2015-0275175-A1). Note thatanti-coagulants were not added to the PBS media used for formulation ofthe final early apoptotic cell dose for infusion.

Summary

The objective of this study was to produce a stable, high yield earlyapoptotic cell population. The rational for use of anticoagulants wasthat aggregates were seen first in patients with high-triglycerides, butlater in a significant portion of other patients. A concern here was thedisclosure in U.S. Pat. No. 6,489,311 that the use of anticoagulantsprevented cell apoptosis.

In short, with minimal impact on the composition, viability, stability,and the apoptotic nature of the cells, there was a significantimprovement of at least 10-20% in the number of collected cells in thefinal product (Yield) when anticoagulant was added. In this study an upto 13% increase in yield was shown, which represents 26.8% augmentationin yield in controlled conditions but in real GMP conditions it went upto 33% and more augmentations in cell number then can be produced in asingle collection. This effect is crucial, since it may avoid the needfor a second apheresis from a donor.

This effect was surprising because the anticipated impact was expectedto be dissolution of mild aggregates. It had been hypothesize thatthawing cells with anticoagulant reduced the amount of aggregates. Whenformed, these aggregates eventually lead to massive cell loss. Cellscollected and frozen without anticoagulant demonstrated aggregateformation at thawing, immediately after wash. Furthermore, a high levelof aggregates was also detected in cells that were frozen withoutanticoagulant and resuspended with media containing anticoagulant. Noaggregates were seen in cells that were both frozen and resuspended withmedia containing anticoagulant. Taken together, it was conclude that theaddition of anticoagulants during freezing and apoptosis induction is ofhigh importance, and did not appear to negatively impact the inductionof early apoptosis on the cell population.

Recovery of early apoptotic cells was further tested, for example,following 24 hours of storage at 2-8° C., for stability purposes, duringwhich an average cell loss of 3-4.7% was measured, regardless ofmanufacturing conditions, with favorable results for cells that wereboth frozen and thawed with media containing anticoagulant (0.2±0.4%cell loss following 24 hours of FP storage), suggesting that addition ofanticoagulant is critical during freezing and thawing, but once finallyformulated, the early apoptotic cell population is stable. Extended timepoint studies showed this stability to at least 72 hours.

Apoptosis and viability, as well as cell composition of the FP productwere not significantly affected by the addition of anticoagulant at thefreezing and/or thawing stage. Values measured from a wide variety ofcharacteristics were similar, indicating the ACDhep did not change theearly apoptotic cell characteristics and the final product met theacceptance criteria of ≥40% apoptotic cells.

The assay used to test apoptotic cells potency was based on immaturedendritic cells (iDCs), DCs that are characterized by functions such asphagocytosis, antigen presentation, and cytokine production.

The HLA-DR (MHC class II) membrane molecule and co-stimulatory moleculeCD86 were selected as markers to detect the tolerogenic effects ofantigen-presenting cells (APCs). Using flow cytometry, changes inexpression of HLA-DR and CD86 on iDCs were measured followingstimulation with LPS, as well as in the presence of the early apoptoticcell population manufactured with- or without anticoagulant andstimulated with LPS. Early apoptotic cell populations were offered toDCs in ascending ratios of 1:2, 1:4, and 1:8 iDCs: early apoptotic cellpopulation. As presented in Table 6, it was shown that early apoptoticcell population enhanced the tolerogenic effect over stimulated DCs in adose-dependent manner, with slightly better results for early apoptoticcell population manufactured with anticoagulant both at freezing andapoptosis induction.

Taken together, it was concluded that addition of anticoagulant to bothfreezing and apoptosis media is of high importance to increase cellrecovery and avoid massive cell loss due to aggregates, and to avoid inmany cases a second round of apheresis from a donor. It was shown thatall cells met acceptance criteria for the validated FP, indicating thatthe addition of anticoagulant does not impair the FP.

Example 2: Effect of Apoptotic Cells on Cytokine Release in an In VitroCytokine Storm Model

Objective:

Test the effect of apoptotic cells on the level of cytokine stormmarkers (cytokines IL-6, IL-10, MIP-1α, IL-8, TNF-α, MIP-1β, MCP-1, andIL-9) in a cytokine storm induced in an LPS-Sterile model of macrophageactivation syndrome.

Methods:

Cell Lines and Culturing Reagents

The human lymphoma cell line Raji (eCACC, UK, access no. 85011429), thehuman cervical adenocarcinoma cell line HeLa (ATCC, USA, number: CCL-2)and HeLa-CD19 (ProMab, USA, cat. no. PM-Hela-CD19) were cultured in RPMI1640 (Gibco, ThermoFisher Scientific, USA, cat. no. 31870-025)supplemented with 10% FBS (Gibco, ThermoFisher Scietific, South America,cat. no. 12657-029), 2 mM GlutaMAX (Gibco, ThermoFisher Scientific, USA,cat. no. 35050-038), and 100 U/ml Penicillin+100 U/ml Streptomycin(Gibco, ThermoFisher Scientific, USA, cat. no. 15140-122), henceforthreferred to as “Complete Medium”. HeLa-CD19 medium was furthersupplemented with 1 μg/ml puromycin (Sigma-Aldrich, USA, cat. no.P9620), as the selective antibiotics, during standard culturing.

All cells were kept in sub-confluent conditions. Raji cells weremaintained in a concentration range of 0.3×10⁶-2×10⁶ cell/ml. HeLa andHeLa-CD19 cells were passaged when receptacle was filled to 90%confluence.

Primary monocytes were isolated from blood donations buffy coats (ShebaMedical Center, Israel). First, peripheral blood mononuclear cells(PBMCs) were isolated on a Ficoll density gradient (Ficoll-Paque PLUS,GE Healthcare, UK, cat. no. 17-1440-03). Upon centrifugation (800×g,2-8° C., 20 min. with break 0), the interphase containing the PBMCs weretransferred to a fresh test tube and washed with RPMI-1640 (Lonza,Switzerland, cat. no. BE12-918F) supplemented with 2 mM L-glutamine(Lonza, Switzerland, cat. no. BE17-605E) and 10 mM Hepes (Lonza,Switzerland, cat. no. BE17-737B), henceforth “Wash Medium”, andcentrifuged (650×g, 2-8° C., 10 min.). Pelleted cells were re-suspendedin “Wash Medium” to a concentration of 15×10⁶ cell/ml. Cells were seededas a 0.9 ml drop at the center of a 35-mm plate (Corning, USA, cat. no.430165). Plates were incubated for 1.5 h in a humidified incubator (37°C., 5% CO₂), allowing monocytes to adhere, and then washed three timeswith pre-warmed PBS (Lonza, Switzerland, cat. no. BE17-516F), removingother cell types. After washing, cells were cultured in 2 ml RPMI 1640(Gibco, ThermoFisher Scientific, USA, cat. no. 31870-025) supplementedwith 10% FBS (Gibco, ThermoFisher Scietific, South America, cat. no.12657-029), 2 mM GlutaMAX (Gibco, ThermoFisher Scientific, USA, cat. no.35050-038), and 100 U/ml Penicillin+100 U/ml Streptomycin (Gibco,ThermoFisher Scientific, USA, cat. no. 15140-122), aka “CompleteMedium”.

All cell lines were cultured in a humidified incubator at 37° C. andcontaining 5% CO₂.

In brief, and following manufacturer's guidelines, target cells (HeLa orHeLa-CD19) were cultured alone or in conjunction with monocytes. Aftertarget cells adhered to the plate (6 h-overnight), cultures were exposedto y×10⁶ ApoCells cells for 1 h, after which these cells were washed offby 4-5 washes of RPMI. Removal of ApoCells cells was confirmed visuallyunder a light microscope. 10 ng/ml LPS (Sigma-Aldrich, USA, cat. no.L4391) was introduced to the co-culture and incubated for 1 h. Afterincubation, LPS was removed by 3-5 washing cycles with RPMI. ViableCD19-CAR T cells or naïve T cells were added at the designated E/Tratio(s) and incubated for 4 h. To collect media, plates werecentrifuged at 250×g, 2-25° C., 4 min. (Centrifuge 5810 R, Eppendorf,Germany) to sediment cells. 50 μl of supernatant medium from each wellwas transferred to a fresh flat-bottom 96-well microplate well (Corning,USA, cat. no. 3596) and 50 μl CytoTox 96 Reagent was added to each well.Plates were incubated in the dark at room temperature for 30 min., afterwhich the reaction was terminated by addition of 50 μl Stop Solution perwell. Absorbance was read at 492 nm using Infinite F50 (Tecan,Switzerland) and captured using Magellan F50 software. Data analysis andgraph generation was performed using Microsoft Excel 2010.

Analysis of cytokine release was performed using Liminex technologyfollowing incubation with apoptotic cells or incubation with supernatantfrom apoptotic cells.

Results:

FIGS. 4A through 4H show that there was a significant reduction in thelevels of cytokine storm markers IL-10, IL-6, MIP-1α, IL-8, TNF-α,MIP-1β, MCP-1, and IL-9 which were induced by LPS in an in vitro modelof macrophage activation syndrome. While administration of ApoCells toachieve a macrophage:Apocell ratio of 1:8 resulted in significantlydecreased levels of both IL-10, IL-6, MIP-1α, IL-8, TNF-α, MIP-1β,MCP-1, and IL-9 released into the medium (FIGS. 4A, 4B, 4C, 4D, 4E, 4F,4G, 4H), administration of ApoCells to achieve a macrophage: ApoCellratio of 1:16 actually inhibited or nearly inhibited the release ofcytokines IL-10, IL-6, MIP-1α, IL-8, TNF-α, MIP-1β, MCP-1, and IL-9 inthis model.

Addition of apoptotic cells resulted in the inhibition of at least 20pro-inflammatory cytokine and chemokines induced in macrophageactivating, a sample of the results are shows in FIGS. 4A-4H. The commonmechanism for pro-inflammatory cytokine and chemokine release is NF-κBinhibition.

The inhibition of release of pro-inflammatory cytokines and chemokinesappears to be specific, as examination of cytokine IL-2R (IL-2 receptor)levels under similar conditions showed that IL-2R levels released wasnot influenced in the same manner at the pro-inflammatory cytokines.(FIG. 4I). Addition of apoptotic cells increased the release of IL-2R at1:4 and 1:8 ratios. Further, FIG. 4J shows that apoptotic cells had noinfluence on release of IL-2 over a 24 hour time period. Activation ofthe IL-2 receptor is considered to have an essential role in keyfunctions of the immune system including tolerance.

Conclusion:

Addition of early apoptotic cells in a cytokine storm model ofmacrophage activation syndrome in the presence of cancer and CAR-19,resulted in significant reduction and, surprisingly even prevention ofpro-inflammatory cytokines, for example IL-10, IL-6, MIP-1α, IL-8,TNF-α, MIP-1β, MCP-1, and IL-9, while increasing or not affectingcytokine IL-2R levels. Thus, the results here show that whilepro-inflammatory cytokines were reduced by incubation with apoptoticcells, IL-2 and IL-2R were not influenced in the same manner withincubation of early apoptotic cells. Thus, the T-cell associatedcytokines are not influenced by the CAR T-cell therapy+apoptotic cells,whereas the innate immunity cytokines, for example those released frommonocytes, macrophages, and dendritic cells are.

Example 3: Effect of Apoptotic Cells on Cytokine Storm without aNegative Effect on the CAR-T Cell Efficacy

Objective:

Test the effect of apoptotic cells or supernatants derived fromapoptotic cells on cytokine storm marker cytokines and CAR T-cellefficacy on tumor cells.

Methods:

T4+ CAR T-cells

A solid tumor model (van der Stegen et al., 2013 ibid) reported toinduce cytokine storms in mice was utilized. In this model, T cells wereengineered with a chimeric antigen receptor (CAR) targeting certain ErbBdimers (T4⁺ CAR-T cells), which are often highly up-regulated inspecific solid tumors such as head and neck tumors and ovarian cancers.T-cells were isolated from PBMC separated from peripheral blood usingCD3 micro-beads. Vectors containing the chimeric T4+ receptor wereconstructed and transducer into the isolated T-cells, resulting in T4+CAR T-cells. For the experiments performed herein, T4+ CAR T-cells werepurchased from Creative Biolabs (NY USA) or Promab Biotechnologies (CAUSA). FIG. 5 presents flow cytometry curves verifying the surfaceexpression of 4αβ chimeric receptor on the T4+ CAR T-cells using ananti-CD124 monoclonal antibody (Wilkie et al., ibid). In addition, a PCRprocedure was performed and verified the presence of the vector intransduced T cells.

SKOV3-Luc Cells

SKOV3-luc ovarian adenocarcinoma tissue culture cells were purchasedfrom Cell BioLabs (cat. #AKR-232). SKOV3-luc highly express ErbBreceptors and are a target for the T4⁺ CAR-T cells (van der Stegen etal., 2013, ibid). These cells had been further manipulated toconstitutively express the firefly luciferase gene, allowing tracking ofcell proliferation in vitro and tumor growth and recession in vivo.

Apoptotic Cells

Apoptotic cells were prepared as per Example 1.

Apoptotic Cell Supernatants

Eight (8) million apoptotic cells per seeded per well in a 12-wellplate. After 24 hours the cells were centrifuge (290 g, 4 degreesCelsius, 10 minutes). Supernatant was collected and frozen in aliquotsat −80 degrees until use. Different numbers of cells are used to makesupernatants. Some aliquots contain concentrated supernatants.

Monocyte Isolation

PBMCs were isolated using Ficoll (GE healthcare, United Kingdome) fromperipheral blood buffy coat obtained from healthy, eligible donors.Cells were brought to a concentration of 15×10⁶ cells\ml in RPMI1640(Gibco, Thermo Fisher Scientific, MA, USA) and seeded in a 0.9 ml dropin the middle of 35 mm plates (Corning, N.Y., USA). Plates were thenincubated at 37° C. in 5% CO₂ for 1 hour. At the end of incubation,cells were washed three times with PBS (Biological industries, BeitHaemek, Israel) and adhesion was determined using a light microscope.Cells were then incubated with complete media (RPMI1640+10% heatinactivated FBS+1% Glutamax+1% PenStrep, all from Gibco).

An alternative method of monocyte isolation was also used wherein humanmononuclear cells were isolated from heparinized peripheral blood bydensity gradient centrifugation. The isolated mononuclear cells thenwere separated into monocyte, B-cell and T-cell populations bypositively selecting monocytes as the CD14+ fraction by magnetic beadseparation (Miltenyi Biotec., Auburn, Calif., USA), positively selectingB-cells as the CD22+ fraction, and negatively selecting T-cells as theCD14-CD22− fraction. Purity was greater than 95 percent for monocytes.

For macrophage differentiation, at the end of adhesion, cells werewashed three times with PBS then incubated with RPMI1640+1% Glutamax+1%PenStrep and 10% heat inactivated human AB serum (Sigma, MO, USA). Cellswere incubated at 37° C. and 5% for 7-9 days, with media exchange at day3 and day 6. Differentiation was determined by morphology via lightmicroscope.

Supernatant from Apo+ Monocytes

CD14+ monocytes were cultured with apoptotic cells as prepared above ata ratio of 1:16, for 24 h. The number of monocytes was: 0.5 millioncells per well in a 12-well plate and the number of apoptotic cells was:8 million cells per well in a 12-well plate. After incubation for 24hours the cells were centrifuge (290 g, 4 degrees Celsius, 10 minutes).Supernatant was collected and frozen in aliquots at −80 degrees untiluse. Similar procedures could be performed at different ratios ofmonocytes:apoptotic cells and/or using other sources of cells, such asmacrophages and dendritic cells.

In Vitro Culturing Conditions

Initial experiments were performed by incubating SKOV3-luc cancer cellswith apoptotic cells, or apoptotic supernatants, for 1 hour followed byco-culturing with T4+ CAR T-cells (+/−monocytes-macrophages) for 48hours.

In order to simulate in vivo conditions, 1×10⁵ THP-1 cells/ml (HTCCUSA), or monocytes or macrophages or dendritic cells, will bedifferentiated with 200 nM (123.4 ng/ml) phorbol myristate acetate (PMA)for 72 hrs and will then be cultured in complete medium without PMA foran additional 24 h. Next, cancer or tumor cells—for example SKOV3-luccells will be plated in a 24-well plate at 5×10⁵ SKOV3-luc cells/well onthe differentiated THP-1 cells. Following initial culturing of thecancer or tumor cells, 4×10⁵-8×10⁵ apoptotic cells (ApoCell) will beadded to the culture for 1-3 h to induce an immunotolerant environment.The ratio of cancer cell to ApoCell will be optimized for each celltype. After washing, the co-culture will be treated with 10 ng/ml LPSafter which 1×10⁶ T4⁺ CAR T cells (or a quantity to be determined by aneffector/target (E/T) ratio graph) will be added. The ratios of tumorcells and T4+ CAR T-cells will be varied in order to generateeffector/target (E/T) ratio graphs for each tumor or cancer cell type.

To assay for SKOV3 cancer cell cytotoxicity, lysates were prepared andluciferase activity was determined after the 48 hour incubation period.Additional experiments will be performed assaying for cancer or tumorcell cytotoxicity for the other cancer cell types and at intervalswithin the 48 h incubation time period. Alternatively, Promega's CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega, cat #G1780) will beused.

Lysate Preparation

SKOV3-luc cell lysates were prepared by washing the SKOV3-luc monolayerwith PBS to remove any residual serum and adding 70 μl CCLR lysis bufferxl/well (for 24-well plates). Detachment was further enhanced byphysical scraping of well bottoms. Following vortexing for 15 seconds,lysates were centrifuged at 12,000 g for 2 minutes at 4° C. Supernatantswere collected and stored at −80° C.

In Vitro Luciferase Activity

To detect luciferase activity in SKOV3-luc cells in culture, LuciferaseAssay System (Promega, cat. #E1501) was used. Calibration of this kitwith the luminometer reader (Core Facility, Faculty of Medicine, EinKerem, Hebrew University of Jerusalem) was done by using QuantiLumrecombinant luciferase (Promega, cat. #E170A). 612 ag-61.2 μg(10⁻²⁰-10⁻⁹ moles) was used to determine detection range and followingmanufacturer's guidelines. In brief, each rLuciferase quantity in 20 μlvolume was placed in a well of black 96-well plates (Nunc). Eachquantity was done in triplicate. 100 μl LAR (luciferin substrate fromLuciferase Assay System kit) was added to each well and read immediatelywith a 10 second exposure.

For luciferase activity reading, lysates were thawed on ice and 20 μlsamples were placed in 20 a black 96-well plate (Nunc). Each sample wasread in duplicate. 100 μl LAR was added and luminescence was read for 10second exposure period every 2.5 minutes for 25 minutes and every 40seconds for the ensuing 10 minutes.

Cytokine Analysis

Initial assays for IL-2, IL-2 receptor (IL-2R), IL-6, IL-1α, IL-4, IL-2,TNF-α were performed. To assay for cytokine release reduction of IL-2,IL-2 receptor (IL-2R), IL-6, IL-1α, IL-4, IL-2, TNF-α as well as othercytokines, supernatants were be collected and examined for selectedcytokine using Luminex MagPix reader and ELISA assays.

Results:

SKOV3-Luc Growth

SKOV3-luc growth was followed using luciferase activity as an indicator,to determine target SKOV3-luc cell number in future experiments.3.8×10⁴-3.8×10⁵ SKOV3-luc cells/well were plated in 24-well plates(Corning) and luciferase activity was monitored daily for 3 days.1.9×10⁵ cells/well or higher cell number plated reach confluence andpresent growth saturation indicated by luciferase activity 2 days afterplating (FIG. 6 ). Note that 3.8×10⁴-1.1×10⁵ SKOV3-luc cells/well werestill in the linear or exponential growth phase three days after plating(FIG. 6 , plots orange, turquoise and purple). Negative control (3.8×10⁵SKOV3-luc cells without LAR substrate) displayed only background-levelreading and demonstrates that bioluminescent readings from SKOV3-luccells result from luciferase activity.

Verification of T4⁺ CAR-T Cell Activity Against SKOV3-Luc Tumor Cells

To corroborate the T4⁺ CAR-T cell activity, monolayers of SKOV3-luc wereexposed to either 1,000,000 (one million) T4⁺ CAR-T cells or to1,000,000 (one million) non-transduced T cells. After 24 h incubation,T4⁺ CAR-T cells reduced SKOV3-luc proliferation by 30% compared to thenon-transduced T cell control (FIG. 7 ), showing anti-tumor activity ofthe T4⁺ CAR-T cells.

Activity of Stand-Alone T4+ CAR-T Cells Against SKOV3-Luc Tumor Cellswas Compared to Activity Post Exposure to Apoptotic Cells

Apoptotic cells (ApoCell) and apoptotic cell supernatants (ApoSup andApoMon Sup) were tested to determine if they interfere with T4+ CAR-Tcell anti-tumor activity. The SKOV3-luc tumor cells were incubate withApoptotic Cells for one hour, followed by the addition of T4+ CAR-Tcells (500,000, five hundred thousands) or T4+ non-transduced T cells(500,000, five hundred thousands) (ratio of 1:2 T4⁺ CAR-T cells toApoptotic Cells). The tumor cell/Apoptotic cell/T4⁺ CAR T-cells werethen co-cultured for 48 h. The control SKOV3-luc tumor cells wereco-cultured with T4+ CAR-T cells and Hartman solution (the vehicle ofApoptotic Cells), but without Apoptotic Cells, for 48 h.

The results showed that after 48 h incubation, T4+ CAR-T cellsanti-tumor activity was superior to incubation with non-transduced Tcells. Similar incubations were performed with apoptotic cells orapoptotic cell supernatants. Surprisingly, T4+ CAR T-cell anti-tumoractivity was comparable with or without exposure to apoptotic cells orapoptotic cell supernatants. (FIG. 8 ).

Effect of Apoptotic Cells on Amelioration, Reduction or Inhibition ofCytokine Storms Resulting from CAR-T Treatment

The effect of apoptotic cells to reduce cytokine storms was examinednext. IL-6 is a prototype pro-inflammatory cytokine that is released incytokine storms (Lee D W et al. (2014) Blood 124(2): 188-195) and isoften used as a marker of a cytokine storm response.

Cultures were established to mimic an in vivo CAR T-cell therapyenvironment. SKOV3-luc tumor cells were cultured in the presence ofhuman monocyte-macrophages and T4+ CAR T-cells. The concentration of11-6 measured in the culture media was approximately 500-600 μg/ml. Thisconcentration of IL-6 is representative of a cytokine storm.

Unexpectedly, IL-6 levels measured in the cultured media of SKOV3-luctumor cells, human monocyte-macrophages, T4+ CAR-T cells, wherein thetumor cells had been previously incubated with apoptotic cells for onehour (ratio of 1:2 T4+ CAR-T cells to Apoptotic Cells) were dramaticallyreduced. Similarly, IL-6 levels measured in the cultured media ofSKOV3-luc tumor cells, human monocyte-macrophages, T4+ CAR-T cells,wherein the tumor cells had been previously incubated with apoptoticcell supernatants for one hour, were also dramatically reduced. Thisreduction in concentration of IL-6 is representative of a decrease inthe cytokine storm (FIG. 9 ).

It was concluded that unexpectedly, apoptotic cells and apoptoticsupernatants do not abrogate the effect of CAR-T cells on tumor cellproliferation while at the same time they down regulatingpro-inflammatory cytokines such as IL-6, which was been described as amajor cytokine leading to morbidity.

Analysis Using a Wider Range of Cytokines

To further evaluate the effect on a possible wider range and levels ofcytokines that are not generated during experimental procedures but doappear in clinical settings during a human cytokine storm, LPS (10ng/ml) was added to the SKOV3-luc culture conditions outlined above. Theaddition of LPS is expected to exponentially increase the cytokine stormlevel. As expected, the addition of LPS increased the cytokine stormeffect and as a result IL-6 levels increased to approximately 30,000μg/ml. Other cytokines known to be expressed in high levels during acytokine storm showed elevated levels, for example: TNF-α (250-300μg/ml), IL-1β (200-300 μg/ml), IL1-alpha (40-50 μg/ml) and IL-18 (4-5μg/ml). As shown in FIG. 10 , exposure to apoptotic cells dramaticallyreduced the levels of IL-6 even during the exponential state of thecytokine storm to almost normal levels that may be seen in clinicalsettings, and is not always seen in experimental procedures with CART-cells. This effect was similar across the other pro-inflammatorycytokines TNF-alpha□ IL□□□□□IL□□alpha, IL-1β, □and IL-18, which showed areduction of between 20-90%. Similar results were found when usingapoptotic cell supernatants in place of the apoptotic cells.

Effect of Apoptotic Cells on IL-2 and IL-2R

The concentration of IL-2 measured in culture supernatants followingincubation of SKOV3-luc cells with T4+ CAR T-cells was 1084 μg/ml.Surprisingly, when SKOC3-luc cells were first incubated with apoptoticcells and then T4+ CAR T-cells the concentration of IL-2 increased to1190 μg/ml. Similarly, the concentration of IL-2R measured in culturesupernatants following incubation of SKOV3-luc cells with T4+ CART-cells was 3817 μg/ml. Surprisingly, when SKOC3-luc cells were firstincubated with apoptotic cells and then T4+ CAR T-cells theconcentration of IL-2R increased to 4580 μg/ml. In SKOV3-luc alone theconcentration of Il-2 was 3.2 μg/ml and with the addition of apoptoticcells the concentration was 10.6 μg/ml. In SKOV3-luc alone theconcentration of Il-2R was 26.3 μg/ml and with the addition of apoptoticcells the concentration was 24.7 μg/ml.

Conclusion

CAR-T cell therapy has been documented to cause cytokine storms in asignificant number of patients. These results demonstrate that apoptoticcells and apoptotic cell supernatants surprisingly decreased cytokinestorms cytokine markers without affecting CAR-T cell efficacy againsttumor cells. Moreover, it appears that apoptotic cells increase cytokineIL-2, which may increase duration of CAR T-cell therapy by maintainingor increasing CAR T-cell proliferation.

Example 4: Apoptotic Cell Therapy Prevents Cytokine Storms in MiceAdministered CAR T-Cell Therapy

Objective:

Test the in vivo effect of apoptotic cells or apoptotic cellsupernatants in a solid tumor model (SKOV3 ovarian adenocarcinoma), inorder to determine T4+ CAR T-cell efficacy and the level of cytokinestorm marker cytokines.

Materials and Methods

In Vitro Studies

In vitro methods including methods of making, culturing, and analyzingthe results described above and relevant for use of T4+ CAR T-cells thatrecognize the ErbB target antigen (referred to herein as “T4+ CART-cells”, SKOV3-luc cells, apoptotic cells, apoptotic supernatants,monocytes, macrophages, and the various assays, have all been describedabove in Example 1. The same methods were used herein.

In Vivo Studies

Mice

7-8 week old SCID-beige mice and NSGS mice were purchased from Harlan(Israel) and kept in the SPF animal facility in Sharett Institute.

SKOV3-luc tumor cells (1×10⁶ or 2×10⁶) are inoculated into SCID beigemice or NSGS mice, by either i.p. in PBS or s.c. in 200 ml Matrigel (BDBiosciences). Tumor engraftment is confirmed by bioluminescence imaging(BLI) at about 14-18 days post injection, and mice are sorted intogroups with similar signal intensity prior to T-cell administration.

Mice will receive 30×10⁶ apoptotic cells either 24 hours prior toadministration of T4+ CAR T-cells or concurrent with administration ofT4+ CAR T-cells (10-30×10⁶ T4+ CAR T-cells). Tumor growth will befollowed by bioluminescence imaging (BLI) and circulating cytokinelevels will be determined by Luminex.

In Vivo Luciferase Assay

Tumor growth was monitored weekly through firefly luciferase activity.In brief, 3 mg D-luciferin (E1605. Promega, USA)/mouse (100 μl of 30mg/ml D-luciferin) was injected i.p. into isoflurane-anesthetized miceand ventral images were acquired 10 minutes after injection using IVISImaging System and Live Image image capture software (both from PerkinElmer, USA).

Image acquisition parameters were chosen for each image session byimaging mice that received 0.5×10⁶ SKOV3-luc cells/mouse, 5 minutes postD-luciferin injection the “auto” option. Capture parameters were set forbinning 4, F/stop 1.2 and exposure of 2-4 minutes using the 24× lens.Data analysis and quantification was performed with the Live Imagesoftware and graphs were generated using Microsoft's Excel program.

In Vivo Cytotoxicity

To assess in vivo toxicity of T-cells, organs are collected from mice,formalin fixed, and subjected to histopathologic analysis.

Cytokine Analysis

Supernatants and sera are analyzed using Luminex MagPix reader and/orELISA kits, cytometric bead arrays (Th1/Th2/Th17; BD Biosciences) asdescribed by the manufacturers. For example, analysis may be forpro-inflammatory cytokine, which in one case would be IL-6, though, insome embodiments, any of the cytokines listed in Tables 1 and 2 or knownin the art may be analyzed herein.

Results

Calibrating SKOV3-Luc Tumors In Vivo

0.5×10⁶, 1×10⁶ or 4.5×10⁶ SKOV3-luc cells were injected i.p. to SCIDbeige mice and bioluminescence imaging (BLI) was conducted weekly inorder to follow tumor growth, as described in the Methods (data notshown).

Clinical Score of Mice

Mice displayed no clinical symptoms for the initial 4 weeks. However, 28days post SKOV3-luc injection, the mice that received the high dose(4.5×10⁶; purple line) began to lose weighed steadily (FIG. 11A) and theoverall appearance of the mice deteriorated, manifested in lethargy,abnormal pacing and general loss of activity. This group was culled atthe day 39, and an abdominal autopsy was performed to expose tumorappearance and size (FIG. 11B). SKOV3-luc tumors were large, solid,vascularized and displayed a whitish shining complexion. One large tumorpredominated on the side of the injection (left) either caudal orrostral in the abdominal cavity. This tumor encompassed approximately25-75% of the cavity and clearly pressed and disturbed the intestines.Smaller foci were also observed at various locations within theabdominal cavity. Tumors were contained within the abdominal cavity andno other tumors were observed in any other part of the body in any mice.Mice receiving low (0.5×10⁶) or medium (1×10⁶) dose of SKOV3-luc ceasegaining weight 40 days after SKOV3-luc injection and began to steadilylose weighed. Experiment was terminated 50 days after SKOV3-lucinjection.

SKOV3-Luc Tumor Kinetics

PBS was injected to control for SKOV3-luc cells and these mice did notexhibit any luciferase activity throughout the experiment (FIG. 12 ,Left panel). Tumor detection and growth was dose-dependent. Lower dose(0.5×10⁶ SKOV3-luc cells) began to display tumors 25 days post-injection(4/5 animals), medium dose (1×10⁶) injections showed tumors at 18 dayspost-injection (4/5 animals), whereas at higher dose (4.5×10⁶) tumorswere detected as early as 11 days post-injection in 3/5 animals and byday 18 all animals displayed well-established tumors (FIG. 12 and FIGS.13A-13D).

CAR T-Cell Therapy Induces Cytokine Release Syndrome

Three groups of tumor-free mice as well as mice with tumors areadministered (i.p. or directly into the tumor) increasing doses of T4+CAR T-cells (3×10⁶, 10×10⁶ or 30×10⁶). At the highest dose, tumor-freemice and mice with tumors demonstrate subdued behavior, piloerection,and reduced mobility within 24 h, accompanied by rapid weight lossfollowed by death within 48 hrs. At least Human interferon-gamma andmouse IL-6 are detectable in blood samples from the mice given thehighest dose of CAR T-cells. Animals that receive a high dose of CART-cells directed to a different tumor antigen do not exhibit weight lossor behavioral alterations.

Administration of Apoptotic Cells Inhibits or Reduces the Incidence ofCytokine Release Syndrome Induced by CAR T-Cell Therapy

One group of mice given the highest dose of CAR T-cells is concomitantlyadministered 2.10×10⁸/kg apoptotic cells, which was previouslydemonstrated to be a safe and effective dose. Mice receiving human CART+ apoptotic cells have significantly lowered levels of mouse IL-6,lower weight loss, and reduced mortality.

Example 5: Effect of Combination Immune Therapy on In Vitro DiffuseTumor Models

Objective:

Test the effect of apoptotic cells or supernatants derived fromapoptotic cells in a diffuse tumor model where the cancer is widelyspread and not localized or confined, in order to determine CAR T-cellefficacy on the cancer cells and the level of cytokine storm markercytokines.

Methods:

CD19+ T4+ CAR T-Cells (“CD19+ CAR T-Cells”)

CD19-specific CAR-T cells were purchased from ProMab (Lot #012916). TheT cells were 30% positive for CAR (according to manufacturer's FACSdata—Fab staining). Briefly, cells were thawed into AimV+5%heat-inactivated FBS, centrifuged (300 g, 5 minutes, room-temperature),and resuspended in AimV. On day 6 of the experiment 20×10⁶ cells wereinjected IV per mouse (70% AnnexinPI negative, of which 30% CARpositive).

Recombinant HeLa cells expressing CD19 will be used as a controlcell-type that also expresses CD19 on their cell surface.

CD123+ CAR T-Cells

T4+ CAR T-cells will also be engineered with a CAR targeting CD123epitopes (referred to herein as “CD123+ CAR T-cells”).

Raji cells, CD19 expressing HeLa cells, and CD123 expressing leukemiccells

Raji cells Raji cells were purchased from ECACC (Cat. #: 85011429), androutinely cultured in complete medium (RPMI-1640 supplemented with 10%H.I. FBS, 1% Glutamax, 1% Penicillin/Streptomycin), and maintained at aconcentration of 3×10⁵-3×10⁶ cells/ml. On day 1 of the experiment0.1×10⁶ cells were injected IV per mouse.

Similarly, CD19 expressing HeLa cells will be generated in thelaboratory and used as a target for CD19+ CAR T-cells. CD123 expressingleukemic cells will be used as targets for CD123+ CAR T-cells. Inaddition, primary cancer cells will be utilized as a target for CART-cells.

HeLa cells expressing CD19 were prepared using methods known in the art.Cells will be cultured as is well known in the art.

CD123 is a membrane biomarker and a therapeutic target in hematologicmalignancies. CD123 expressing leukemic cells, for example leukemicblasts and leukemic stem cells will be cultured as is known in the art.

Apoptotic cells, Apoptotic cell supernatants and monocyte isolation,will be prepared as described in Example 1. Early apoptotic cellsproduced were at least 50% annexin V-positive and less than 5%PI-positive cells.

Macrophages.

Were generated from CD14 positive cells by adherence.

Dendritic Cells.

Were CD14 derived grown in the presence of IL4 and GMCSF.

Flow-Cytometry.

The following antibodies were used:hCD19-PE (eBiosciences, Cat.#12-0198-42); mlgGI-PE (eBiosciences, Cat. #12-0198-42); hCD3-FITC(eBiosciences, Cat. #11-0037-42); mIgG2a-FITC (eBiosciences, Cat.#11-4724-82). Acquisition was performed using FACS Calibur, BD.

Naïve T cells.

Naïve T cells were isolated from Buffy coat using magnetic beads (BD),and cryopreserved in 90% human AB serum and 10% DMSO. Thawing andinjection was identical to the CAR-T cells.

In Vitro Culturing Conditions

Cell Lines and Culturing Reagents

The human lymphoma cell line Raji (eCACC, UK, access no. 85011429), thehuman cervical adenocarcinoma cell line HeLa (ATCC, USA, number: CCL-2)and HeLa-CD19 (ProMab, USA, cat. no. PM-Hela-CD19) were cultured in RPMI1640 (Gibco, ThermoFisher Scientific, USA, cat. no. 31870-025)supplemented with 10% FBS (Gibco, ThermoFisher Scietific, South America,cat. no. 12657-029), 2 mM GlutaMAX (Gibco, ThermoFisher Scientific, USA,cat. no. 35050-038), and 100 U/ml Penicillin+100 U/ml Streptomycin(Gibco, ThermoFisher Scientific, USA, cat. no. 15140-122), henceforthreferred to as “Complete Medium”. HeLa-CD19 medium was furthersupplemented with 1 μg/ml puromycin (Sigma-Aldrich, USA, cat. no.P9620), as the selective antibiotics, during standard culturing.

All cells were kept in sub-confluent conditions. Raji cells weremaintained in a concentration range of 0.3×106-2×106 cell/ml. HeLa andHeLa-CD19 cells were passaged when receptacle was filled to 90%confluence.

Primary monocytes were isolated from blood donations buffy coats (ShebaMedical Center, Israel). First, peripheral blood mononuclear cells(PBMCs) were isolated on a Ficoll density gradient (Ficoll-Paque PLUS,GE Healthcare, UK, cat. no. 17-1440-03). Upon centrifugation (800×g,2-8° C., 20 min. with break 0), the interphase containing the PBMCs weretransferred to a fresh test tube and washed with RPMI-1640 (Lonza,Switzerland, cat. no. BE12-918F) supplemented with 2 mM L-glutamine(Lonza, Switzerland, cat. no. BE17-605E) and 10 mM Hepes (Lonza,Switzerland, cat. no. BE17-737B), henceforth “Wash Medium”, andcentrifuged (650×g, 2-8° C., 10 min.). Pelleted cells were re-suspendedin “Wash Medium” to a concentration of 15×10⁶ cell/ml. Cells were seededas a 0.9 ml drop at the center of a 35-mm plate (Corning, USA, cat. no.430165). Plates were incubated for 1.5 h in a humidified incubator (37°C., 5% CO2), allowing monocytes to adhere, and then washed three timeswith pre-warmed PBS (Lonza, Switzerland, cat. no. BE17-516F), removingother cell types. After washing, cells were cultured in 2 ml RPMI 1640(Gibco, ThermoFisher Scientific, USA, cat. no. 31870-025) supplementedwith 10% FBS (Gibco, ThermoFisher Scietific, South America, cat. no.12657-029), 2 mM GlutaMAX (Gibco, ThermoFisher Scientific, USA, cat. no.35050-038), and 100 U/ml Penicillin+100 U/ml Streptomycin (Gibco,ThermoFisher Scientific, USA, cat. no. 15140-122), aka “CompleteMedium”.

All cell lines were cultured in a humidified incubator at 37° C. andcontaining 5% CO₂.

CD19-CAR T cells (ProMab, USA, cat. no. FMC63) were delivered either inAIM-V medium or frozen. Cryopreserved CAR T cells for in vitroexperiments were thawed on the day of the experiment in a 35-38° C. bathand immediately immersed in pre-warmed AIM V medium (Gibco, ThermoFisherScientific, USA, cat. no. 12055-091) supplemented with 5% FBS (Gibco,South America, cat. no. 12657-029). DMSO was removed by centrifuging thecells (300×g, room temperature, 5 min.) and re-suspending in pre-warmedAIM V medium. Concentration and viability of CD19-CAR+ cell populationwas determined by anti-FLAG (BioLegend, USA, cat. no. 637310) stainingand by Annexin V and PI staining (MEBCYTO Apoptosis kit, MBL, USA, cat.no. 4700) read with FACSCalibur flow cytometer (BD, USA).

For Naïve T cell isolation, PBMCs were extracted either fromleukapheresis fractions collected from informed consenting eligibledonors at Hadassah Medical Center (Ein Kerem Campus, Jerusalem, Israel)using a Cobe Spectra™ apheresis apparatus (Gambro BCT, USA) according toLeaukapheresis Unit's SOP or from buffy coats (Sheba Medical Center,Israel) loaded on a Ficoll density gradient and centrifuged 800×g, 2-8°C., 20 min. T cells were isolated from the positive fraction usingMagniSort Human CD3 Positive Selection Kit (eBioscience, USA, cat. no.8802-6830-74) following manufacturer's guidelines. T cells werecryopreserved in “Complete Medium” (defined above) containing anadditional 20% FBS (Gibco, ThermoFisher Scietific, South America, cat.no. 12657-029) and 5% DMSO (CryoSure-DMSO, WAK-Chemie Medical GmbH,Germany, cat. no. WAK-DMSO-70) and thawed on the day of experimentparallel to the CD19-CAR T cells.

LDH Cytotoxicity Assay

Lactate dehydrogenase (LDH), a stable cytosolic enzyme, is released bycells undergoing lysis in a correlative manner. Hence, LDH levels in themedium can be used to quantify cytotoxic activity. CytoTox 96Non-Radioactive Cytotoxicity Assay (Promega, USA, cat. no. G1780) is acolorimetric assay to quantify LDH levels in the medium. A tetrazoliumsalt substrate (iodonitro-tetrazolium violet, INT) is introduced to themedium in excess and LDH converts the substrate into a red formazanproduct. The amount of red color formed is directly proportional to thenumber of cells lysed.

In brief, and following manufacturer's guidelines, target cells (HeLa orHeLa-CD19) were cultured alone or in conjunction with monocytes. Aftertarget cells adhered to the plate (6 h-overnight), cultures were exposedto y×10⁶ ApoCells cells for 1 h, after which these cells were washed offby 4-5 washes of RPMI. Removal of ApoCells cells was confirmed visuallyunder a light microscope. 10 ng/ml LPS (Sigma-Aldrich, USA, cat. no.L4391) was introduced to the co-culture and incubated for 1 h. Afterincubation, LPS was removed by 3-5 washing cycles with RPMI. ViableCD19-CAR T cells or naïve T cells were added at the designated E/Tratio(s) and incubated for 4 h. To collect media, plates werecentrifuged at 250×g, 2-25° C., 4 min. (Centrifuge 5810 R, Eppendorf,Germany) to sediment cells. 50 μl of supernatant medium from each wellwas transferred to a fresh flat-bottom 96-well microplate well (Corning,USA, cat. no. 3596) and 50 μl CytoTox 96 Reagent was added to each well.Plates were incubated in the dark at room temperature for 30 min., afterwhich the reaction was terminated by addition of 50 μl Stop Solution perwell. Absorbance was read at 492 nm using Infinite F50 (Tecan,Switzerland) and captured using Magellan F50 software. Data analysis andgraph generation was performed using Microsoft Excel 2010.

Flow Cytometry Cytotoxicity Assay

HeLa-CD19 (target) and HeLa (control) cells were pre-stained with 5 NMcarboxyfluorescein succinimidyl ester (CFSE, Life Technologies, USA,cat. no. C1157), mixed together, and plated on either fresh plates or onplates populated with isolated primary monocyte. After target cellsadhere to the plate (6 h-overnight), cultures were exposed to y×10⁶ApoCells cells for 1 h. Plates were washed with RPMI 3-5 times andvisually verified that suspended ApoCells cells were washed off. 10ng/ml LPS was introduced to the co-culture and incubated for 1 h, afterwhich LPS was removed by 3-5 washing cycles with RPMI. Viable CD19-CAR Tcells were then added to the co-cultures as indicated by specific E/Tratio(s) and incubated for 4 h. After incubation, cells were harvestedby adding trypsin-EDTA (Biological Industries, Israel, cat. no.03-052-1B) and incubating for 4 min. at 37° C. To terminate theenzymatic activity, two- to four-fold volume of “complete medium” wasadded. Cells were collected, centrifuged at 200×g for 5 min. at roomtemperature and re-suspended in 100 μl RPMI (Gibco, ThermoFisherScientific, USA, cat. no. 15140-122). Staining ensued first againstanti-CD19 (eBioscience, USA, cat. no. 12-0198-42), incubated in dark for30 min. at room temperature. After centrifugation (290×g, 1 min., 2-8°C.) and re-suspended in 300 μl RPMI, cells were stained againstanti-7AAD (eBioscience, USA, cat. no. 00-6993-50). Analysis was gated on7ADD-negative cells (live cells), where live target cell (HeLa-CD19) andlive control cells (HeLa) was calculated. Percent survival wascalculated by dividing percent live target cells by percent live controlcells. To correct for variation in starting cell numbers and spontaneoustarget cell death, percent survival was divided by the ratio of percenttarget cells to percent control cells cultured without effector cells(CD19-CAR T cells). Finally, percent cytotoxicity was determined bysubtracting the corrected survival percentage from 100%².

Initial experiments are performed by incubating Raji cancer cells withCD19+ CAR T-cells (+/−monocytes-macrophages) for 48 hours in order todetermine optimal ratios of CD19+ CAR T-cells to target Raji cancercells, beginning with 5×10⁴ Raji cells/well in a 96-well plate. Aneffector/target (E/T) ratio plate is constructed based on the results.

Combination immunotherapy experiments are performed by incubating theRaji cancer cells with apoptotic cells, or apoptotic supernatants, for 1hour followed by co-culturing with CD19+ CAR T-cells(+/−monocytes-macrophages) for 48 hours.

In order to simulate in vivo conditions, 1×10⁵ THP-1 cells/ml will bedifferentiated with 200 nM (123.4 ng/ml) phorbol myristate acetate (PMA)for 72 hrs and will then be cultured in complete medium without PMA foran additional 24 h. Next, Raji cancer cells will be plated in a 24-wellplate at 5×10⁵ Raji cells/well on the differentiated THP-1 cells.

Following initial culturing of the Raji cancer cells, 4×10⁵-8×10⁵apoptotic cells (ApoCell) will be added to the culture for 1-3 h toinduce an immunotolerant environment. The ratio of cancer cell toApoCell will be optimized for each cell type. After washing, theco-culture will be treated with a pre-determined number of CD19 CAR-Tcells based on the E/T ratio graph. In certain experiments, 10 ng/ml LPSwill be added to the culture media prior to addition of the CD19+ CART-cells. In other experiments, interferon γ (IFN-γ) will be added to theculture media prior to addition of the CD19+ CAR T-cells. The additionof LPS or IFN-γ is expected to exponentially increase the cytokine stormlevel.

To assay for Raji cancer cell cytotoxicity, lysates are prepared andviability is determined after the 48 hour incubation period. Additionalexperiments will be performed assaying for Raji cell cytotoxicity atintervals within the 48 h incubation time period. Alternatively,Promega's CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, cat#G1780) will be used.

Similar experiments are run with CD19 expressing HeLa cells and CD19+CAR T-cells.

Similar experiments are run with CD123 expressing leukemic cells andCD123+ CAR T-cells.

Cytokine Analysis

Initial cytokine assays examine the levels of MIP1a, IL-4, IL-2, IL-2R,IL-6, IL8, IL-9, IL-10, IL-13, IL-15, INF-γ, GMCSF, TNF-α, in theculture supernatant.

Additional cytokine assays examine the level of cytokines IL-10, IL-1β,IL-2, IP-10, IL-4, IL-5, IL-6, IFNα, IL-9, IL-13, IFN-γ, IL-12p70,GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, IL-15/IL-15R, or IL-7, or anycombination thereof.

Cultures were established to mimic an in vivo CAR T-cell therapyenvironment. Raji Burkett Lymphoma cells were cultured in the presenceof human monocyte-macrophages, LPS and CD19+ CAR T-cells without andwith the addition of apoptotic cells.

Raji cells were incubated in the presence of monocytes and LPS, followedby addition of Naïve T-cells (Raji+Naïve T), CD19+ CAR T-cells(Raji+CART), CD19+ CAR T-cells and apoptotic cells (ApoCell) at a ratioof 1:8 CAR T-cells:ApoCells (Raji+CART+ApoCell 1:8), CD19+ CAR T-cellsand apoptotic cells (ApoCell) at a ratio of 1:32 CAR T-cells:ApoCells(Raji+CAR T+ApoCell 1:32), and CD19+ CAR T-cells and apoptotic cells(ApoCell) at a ratio of 1:64 CAR T-cells:ApoCells (Raji+CART+ApoCell1:64). Concentration measurements were made following GM-CSF and TNF-α(TNF-α).

To assay for cytokine release reduction of IL-6, IL-8, and IL-13, aswell as other cytokines, supernatants will be collected and examined forselected cytokine using Luminex MagPix reader and ELISA assays.Cytokines (mouse or human) may be evaluated by Luminex technology usingMAPIX system analyzer (Mereck Millipore)) and MILIPLEX analysis software(Merek Millipore). Mouse IL-6Ra, MIG (CXCL9) and TGF-β1 were evaluatedby Quantikine ELISA (R&D systems).

Tissue Analysis

Bone marrow and liver were evaluated using flow cytometry andimmunohistochemistry. Upon sacrifice liver and bone marrow werecollected for histopathological analysis. Tissues were fixed in 4%formalin for 48 h at room temperature, and then submitted to the animalfacility at the Hebrew University for processing. Bones were decalcifiedprior to processing. Paraffin sections were stained for Hematoxylin andEosin, and CD19.

IFN-γ Effect

IFN-γ effect is evaluated both by STAT1 phosphorylation and biologicalproducts.

Results:

Calibrating Cell Number for Cytotoxicity Assay

To determine the number of Raji cells to be used in the in vitro model,sensitivity limits of the cytotoxicity assay was assessed. 5×10⁴-20×10⁴Raji cells/well were plated in a 96-well plate, in quadruplicate. Lysiswas performed on one set of quadruplicate to be compared with cells thatare still completely viable. Lysis was momentary, adding the lysissolution immediately prior to centrifugation to simulate partial cellcytotoxicity. Indeed, all cell quantities exhibited readings well aboveviable cells, with the 5×10⁴ cell number producing the greatest relativereading (FIG. 14 ; extrapolation of data). Therefore, subsequentexperiments will be using this cell number as default, unless otherwiserequired by experimental deign.

Verification of CD19⁺ CAR-T Cell Activity Against Raji Burkett LymphomaCells

To corroborate the CD19⁺ CAR T-cell activity, monolayers of Raji cancercells are exposed to either 1,000,000 (one million) CD19⁺ CAR-T cells orto 1,000,000 (one million) non-transduced T cells. After 24 hincubation, CD19⁺ CAR-T cells reduce Raji cancer cell proliferation,showing anti-tumor activity of the CD19⁺ CAR-T cells.

Activity of Stand-Alone CD19+ CAR-T Cells Against Raji Burkett LymphomaCells was Compared to Activity Post Exposure to Apoptotic Cells

Apoptotic cells (ApoCell) and apoptotic cell supernatants (ApoSup andApoMon Sup) are tested to determine if they interfere with CD19+ CAR-Tcell anti-tumor activity. The Raji Burkett Lymphoma cells are incubatewith Apoptotic Cells for one hour, followed by the addition ofCD19+CAR-T cells (500,000, five hundred thousands) or CD19+non-transduced T cells (500,000, five hundred thousands) (ratio of 1:2CD19⁺ CAR-T cells to Apoptotic Cells). The tumor cell/Apoptoticcell/CD19⁺ CAR T-cells are then co-cultured for 48 h. The control RajiBurkett Lymphoma cells are co-cultured with CD19+ CAR-T cells andHartman solution (the vehicle of Apoptotic Cells), but without ApoptoticCells, for 48 h.

The results are showing that after 48 h incubation, CD19+ CAR-T cellsanti-tumor activity was superior to incubation with non-transduced Tcells. Similar incubations will be performed with apoptotic cellsupernatants. Surprisingly, CD19+ CAR T-cell anti-tumor activity iscomparable with or without exposure to apoptotic cells or apoptotic cellsupernatants.

No negative effect of apoptotic cells on CAR-modified T cells againstCD19 both in vitro was seen with comparable E/T ratio results of CAR Tin the presence or absence of apoptotic cells.

Verification of CD19⁺ CAR-T Cell Activity Against HeLa Leukemia Cells

HeLa cells are specific CD19 expressing cells, which renders themsusceptible to CAR CD19⁺ T-cell activity. In addition, in contrast toRaji cells, which are a non-adherent cell line, HeLa cells are adherent.

To corroborate the CD19⁺ CAR T-cell activity, monolayers of HeLa cancercells were exposed to either 1,000,000 (one million) CD19⁺ CAR-T cellsor to 1,000,000 (one million) non-transduced T cells. After 24 hincubation, CD19⁺ CAR-T cells reduce HeLa cancer cell proliferation,showing anti-tumor activity of the CD19⁺ CAR-T cells (FIG. 15 CD19⁺+RPMIand CD19⁺+CAR T-19 cells).

Activity of Stand-Alone CD19⁺ CAR-T Cells Against CD19⁺ HeLa Cells wasCompared to Activity Post Exposure to Apoptotic Cells

Apoptotic cells (ApoCell) were tested to determine if they interferewith CD19+ CAR-T cell anti-tumor activity. The HeLa cells were incubatedwith Apoptotic Cells for one hour, followed by the addition of CD19+CAR-T cells (500,000, five hundred thousand) or CD19+ non-transduced Tcells (Naïve T cells; 500,000, five hundred thousand) (ratio of 1:2CD19⁺ CAR-T cells to Apoptotic Cells). The tumor cell/Apoptoticcell/CD19⁺ CAR T-cells were then co-cultured for 48 h. The control HeLacells were co-cultured with CD19+ CAR-T cells and RPMI (the vehicle ofApoptotic Cells), but without Apoptotic Cells, for 48 h. The CD19⁺ CAR-Tcell: HeLa cell ratio (E/T ratio) ranged from 5-20 (FIG. 15 ).

FIG. 15 shows that after 48 h incubation, CD19+ CAR-T cells anti-tumoractivity was superior to incubation with non-transduced T cells (Naïvecells) or buffer alone. Similar incubations were performed withapoptotic cells. Surprisingly, CD19⁺ CAR T-cell anti-tumor activity wascomparable with or without exposure to apoptotic cells. Similarexperiments are performed using apoptotic cell supernatants. FIG. 15shows the same in vitro cytotoxicity effect of CAR T-CD19 therapy withor without the addition of ApoCells.

No negative effect of the apoptotic cells on CAR-modified T cellsagainst CD19+HeLa cells was observed at comparable E/T ratios in thepresence or absence of apoptotic cells.

Thus, the same in vitro cytotoxic effect of the CD19⁺CAR T-cells wasobserved with or without the addition of early apoptotic cells.

Effect of Apoptotic Cells on Amelioration, Reduction or Inhibition ofCytokine Storms Resulting from CAR-T Treatment

Cytokines IL-8 and IL-13 are measured in the culture media prior to andfollowing addition of CD19+ CAR T-cells and are showing a concentrationconsistent with a cytokine storm. Addition of apoptotic cells orapoptotic cell supernatant is showing a reduction of IL-8 and IL-13concentrations in the media.

Analysis Using a Wider Range of Cytokines

To further evaluate the effect on a possible wider range and levels ofcytokines that are not generated during experimental procedures but doappear in clinical settings during a human cytokine storm, LPS (10ng/ml) was added to the Raji cell culture conditions outlined above inthe presence of cancer and CAR-19. The addition of LPS was expected toexponentially increase the cytokine storm level. Exposure to apoptoticcells is dramatically reduced the levels of cytokines. The resultspresented in FIG. 16 and FIG. 17 show that while addition of CD19+ CART-cell greatly increases cytokine concentration (pg/ml) of GM-CSF andTNF-α in the culture medium, there is a significant decrease of bothGM-CSF and TNF-α in the presence of apoptotic cells. The decrease in thecytokine concentration is dose dependent with respect to apoptotic cellratio of CAR T-cells to apoptotic cells.

Conclusion:

Apoptotic cells were able to down regulate cytokine markers of cytokinestorm associated with CAR T-cell clinical procedures. Significantly, theapoptotic cells did not show an effect on the tumor activity of the CART-cells. Apoptotic cells decreased pro-inflammatory cytokines thatoriginated from innate immunity and inhibit IFN-γ effect without harmingIFN-γ levels and CAR-T cytotoxicity.

Example 6: Apoptotic Cell Therapy Prevents Cytokine Storms in a DiffuseCancer In Vivo Model Administered Car T-Cell Therapy

Objective: Test the in vivo effect of apoptotic cells or supernatantsderived from apoptotic cells in a diffuse tumor model, in order todetermine CAR T-cell efficacy on the cancer cells and the level ofcytokine storm marker cytokines.

Materials and Methods

In Vitro Studies

See methods described in Example 5 for in vitro studies.

Cells and Cell Culture

Raji Burkitt lymphoma cells (Sigma-Aldrich cat. #85011429) were culturedas per the manufacture's guidelines. CD19+ CAR T-cells, cell cultures,apoptotic cells, apoptotic cell supernatants, monocyte isolation, and invitro measurements are as above for Examples. Early apoptotic cellsproduced were least 50% annexin V-positive and less than 5% PI-positivecells.

In Vivo Studies

Mice

7-8 week old SCID beige mice were purchased from Envigo (formerly knownas Harlan). Mice were kept in an SPF free animal facility in compliancewith institutional IACUC guidelines. During the course of theexperiments the mice were monitored daily, and weighted 3 times a week.Mice showing hind limb paralysis were sacrificed. Upon sacrifice bonemarrow and liver were collected for FACS analysis and histologicalprocessing, and sera were frozen at −80° C. for cytokine profiling. Invivo experiments

SCID beige mice (C.B-17/IcrHsd-Prkdc-SCID-Lyst-bg, Harlan, Israel) werehoused in SPF conditions at The Authority for Animal Facilities (AAF),The Hebrew University of Jerusalem (Ein Kerem Campus, Israel) andfollowing the Association for Assessment and Accreditation of LaboratoryAnimal Care (AAALAC). The studies were approved by The Hebrew UniversityEthics Committee for Animal Experiments, and animal suffering wasminimized as possible.

(FIG. 18A) For the disseminating tumor model, 7-8 week female SCID beigemice were injected i.v. with 1×10⁵ Raji cells suspended in 200 μl RPMI(Gibco, ThermoFisher Scientific, USA, cat. no. 15140-122) per mouse (day1). On day 6, mice of pertinent groups were inoculated i.v. with 30×10⁶cells ApoCells in 200 μl Hartmann's solution Lactated Ringer'sInjection, Teva Medical, Israel, cat. no. AWN2324) per mouse. On day 6,mice of relevant groups were inoculated i.v. with 10×10⁶ viable CD19-CART cells or naïve T cells in 200 μl AIM V per mouse. Control micereceived equal volume of RPMI for each treatment.

Mice were examined for clinical indications and weighed twice a week andwere sacrificed upon development of hind limb paralysis. Pathologicalsamples of bone and liver were prepared by the Animal Facility Unit ofThe Hebrew University of Jerusalem and stained against human CD20 (CellMarque, USA, clone L26, cat. no. 120M-84), to detect Raji cells, andagainst human CD3 (Cell Signaling Technology, USA, cat. no. 85061), todetect human T cells.

In certain experiments, LPS will be administered to the animal subjectprior to addition of the CD19+ CAR T-cells. In other experiments,interferon-γ (IFN-γ) will be administered prior to addition of the CD19+CAR T-cells. The addition of LPS or IFN-γ is expected to exponentiallyincrease the cytokine storm level.

Cytokine assays examine the level of cytokines including but not limitedto IL-10, IL-1β, IL-2, IP-10, IL-4, IL-5, IL-6, IFNα, IL-9, IL-13,IFN-γ, IL-12p70, GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, IL-15/IL-15R, orIL-7, or any combination thereof. Cytokines (mouse or human) areevaluated by Luminex technology using MAPIX system analyzer (MereckMillipore)) and MILIPLEX analysis software (Merek Millipore). MouseIL-6Ra, MIG (CXCL9) and TGF-β1 are evaluated by Quantikine ELISA (R&Dsystems).

Tissue Analysis

Bone marrow and liver are evaluated using flow cytometry andimmunohistochemistry. Upon sacrifice liver and bone marrow werecollected for histopathological analysis. Tissues were fixed in 4%formalin for 48 h at room temperature, and then submitted to the animalfacility at the Hebrew University for processing. Bones were decalcifiedprior to processing. Paraffin sections were stained for Hematoxylin andEosin, and CD19.

IFN-γ Effect

IFN-γ Effect is Evaluated Both by STAT1 Phosphorylation and BiologicalProducts.

Results

CAR T-Cell Therapy Induces Cytokine Release Syndrome

Three groups of tumor-free mice as well as mice with tumors areadministered (i.p. or directly into the tumor) increasing doses of CD19+CAR T-cells (3×10⁶, 10×10⁶ or 30×10⁶). At the highest dose, tumor-freemice and mice with tumors demonstrate subdued behavior, piloerection,and reduced mobility within 24 h, accompanied by rapid weight lossfollowed by death within 48 hrs. Human interferon-gamma, and mouse IL-6,IL-8, and IL-13 are detectable in blood samples from the mice given thehighest dose of CD19+ CAR T-cells. Animals that receive a high dose ofCD19+ CAR T-cells directed to a different tumor antigen do not exhibitweight loss or behavioral alterations.

Administration of Apoptotic Cells Inhibits or Reduces the Incidence ofCytokine Release Syndrome Induced by CAR T-Cell Therapy

One group of mice given the highest dose of CD19+ CAR T-cells isconcomitantly administered 2.10×10⁸/kg apoptotic cells, which waspreviously demonstrated to be a safe and effective dose. Mice receivinghuman CD19+ CAR T+apoptotic cells have significantly lowered levels ofat least one mouse pro-inflammatory cytokines, lower weight loss, andreduced mortality.

Administration of Apoptotic Cells in Combination with CAR T-CellAdministration Did not Affect CAR T-Cell Anti-Tumor Activity

FIG. 18B shows that the expected death of SCID mice injected with CD19⁺Raji cells without administration of CD19⁺CAR T-cells was 18-21 days.Forty percent (40%) of the mice who received CD19⁺CAR T-cells survivedto at least day 30 (FIG. 18 dash-dot-dash line and dash-double dot-dashline). The percentage of survivors was independent of the addition ofapoptotic cells (FIG. 18 ). The surviving mice were sacrifice on day 30.

Conclusion: There was comparable survival and no negative effect ofapoptotic cells on CAR-modified T cells against CD19 in vivo.

Significant down regulation (p<0.01) of pro-inflammatory cytokinesincluding, IL-6, IP-10, TNF-α, MIP-1α, MIP-1β was documented. IFN-γ wasnot downregulated but its effect on macrophages and dendritic cells wasinhibited both at the level of phosphorylated STAT1 and IFN-γ-inducedexpression of CXCL10 and CXCL9.

Conclusion:

Apoptotic cells decrease pro-inflammatory cytokines that originate frominnate immunity and inhibit IFN-γ effect without harming IFN-γ levelsand CAR-T cytotoxicity.

Example 7: Apoptotic Cell Therapy Prevents Cytokine Storms in a SolidTumor Cancer In Vivo Model Administered CAR T-Cell Therapy

Objective:

Test the in vivo effect of apoptotic cells or supernatants derived fromapoptotic cells in a solid tumor model, in order to determine CAR T-cellefficacy on the cancer cells and the level of cytokine storm markercytokines.

Materials and Methods

In Vitro Studies

Cells and Cell Culture

CD19+ CAR T-cells, Second generation CAR-T-CD19 cells containing TMCD28were used, cell cultures, apoptotic cells, apoptotic cell supernatants,monocyte isolation, and in vitro measurements were as above for Examples2 & 4 & 6. Early apoptotic cells produced were least 50% annexinV-positive and less than 5% PI-positive cells, as described in detail inExample 1.

In Vivo Studies

Mice

7-8 week old SCID-beige mice and NSGS mice were purchased from Harlan(Israel) and kept in the SPF animal facility in Sharett Institute.

SCID beige mice or NSGS mice were inoculated with CD19 expressing Helacells, that can adhere to the peritoneum, in order to form solidintra-peritoneal tumors. Mice were sorted into groups prior to T-celladministration.

Six days post i.v. inoculation, mice were administered 10×10⁶ CD19+ CART-cells with and without apoptotic cell (ApoCell) preconditioning on day5. Mice receiving pre-conditioning were administered 5×10⁶ or 30×10⁶ApoCells. Tumors were surveyed weekly and circulating cytokine levelswere monitored weekly and determined by the Luminex system. 25 mousecytokines and 32 human cytokines were evaluated using the Luminextechnology. Upon termination of the experiment, mice were culled andorgans (bone marrow, liver and spleen) were examined (by FACS andimmunohistochemistry) for the presence/size of tumors.

Cytokine assays examined the level of cytokines including but notlimited to GM-CSF, IFNγ, IL-1β, IL-10, IL-12p70, IL-13, IL-15, IL-17A,IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, MIP-1α, TNFα, MIP-1β, IFNα, andIP-10. Cytokines (mouse or human) were evaluated by Luminex technologyusing MAPIX system analyzer (Mereck Millipore)) and MILIPLEX analysissoftware (Merek Millipore). Mouse IL-6Ra, MIG (CXCL9) and TGF-β1 wereevaluated by Quantikine ELISA (R&D systems).

Tissue Analysis

Bone marrow and liver are evaluated using flow cytometry andimmunohistochemistry.

IFN-γ Effect

IFN-γ effect was evaluated both by STAT1 phosphorylation and biologicalproducts.

Results

CAR T-cell therapy induces cytokine release syndrome

Three groups of tumor-free mice as well as mice with tumors wereadministered (i.p. or directly into the tumor) increasing doses of CD19+CAR T-cells (3×10⁶, 10×10⁶ or 30×10⁶). At the highest dose, tumor-freemice and mice with tumors demonstrate subdued behavior, piloerection,and reduced mobility within 24 h, accompanied by rapid weight lossfollowed by death within 48 hrs.

FIGS. 19A-19C graphically show the increased levels of IL-6, IP-10 andsurprisingly even TNF-α cytokine release from tumors even before thepresence of CAR T-cells. FIGS. 19A-19C show that unexpectedly IL-6,IP-10, and TNF-α were increased by the presence of cancer cells evenwithout CAR T-cell therapy. In the presence of CAR T-Cell therapy(Hela-CAR T-cell CD-19) the release of cytokines was significantlyaugmented. These results show that the tumor itself releasespro-inflammatory cytokines.

In order to evaluate the benefit of the addition of early apoptoticcells, cytokines GM-CSF, IFNγ, IL-1β, IL-10, IL-12p70, IL-13, IL-15,IL-17A, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, MIP-1α, TNFα, MIP-10, IFNα,and IP-10 were measured in three experiments, wherein the results showedthat macrophage associated cytokines were down-regulated in the presenceof ApoCell administration, while T-cell associated cytokine levels werenot significantly changed (Table 10).

TABLE 10 Cytokine levels from an intra-peritoneum in vivo model thatcontained CD19 expressing Hela cells solid tumor, +/− CAR T-cell CD19therapy, and +/− ApoCell After tumor, Before Tumor After tumor CAR,Pg/ml Car or Apo + CAR + with ApoCell GM-CSF 4 ± 2  88 ± 10 12 ± 4  IFNy4 ± 1  5 ± 8  5 ± 21 IL-1β 8 ± 3 14 ± 6 16 ± 8  IL-10 76 ± 13 222 ± 4436 ± 22 IL-12p70 5 ± 1 188 ± 22 12 ± 11 IL-13 6 ± 2  8 ± 1 8 ± 4 IL-15 4± 2  6 ± 2 8 ± 2 IL-2 4 ± 2 26 ± 2 29 ± 2  IL-4 1 ± 2 16 ± 4 18 ± 6 IL-6 24 ± 6  820 ± 56 74 ± 12 MIP-1α 8 ± 5  99 ± 13 18 ± 8  TNFα 6 ± 2760 ± 33 17 ± 15 MIP-1β 7 ± 1 144 ± 21 21 ± 10 IFNα 74 ± 12  68 ± 26 71± 14 IP-10 8 ± 4 188 ± 33 21 ± 16

Table 10 shows cytokine measurement twenty-four (24) hours after CART-Cell administration+/−ApoCells. Resultant cytotoxicity from CAR T-celltherapy elevated cytokines including GM-CSF, IL-10, IL-12p70, IL-6,MIP-1α, TNFα, MIP-13, and IP-10, the levels of which were significantlydown regulated (p<0.05-0.0001) in the presence of ApoCells. Thesecytokines are mainly associated with macrophages. In contrast, thelevels of cytokines associated with T-cells such as IL-2, IL-4, IL-13,and IL 15 were not changed significantly.

The results presented in FIGS. 19A-C and Table 10, illustrate that theCRS in the context of cancer and CAR has several ingredients: a tumorthat can secrete cytokines; an innate immunity that respond to tumor andto CAR and to other factors; and that CAR T-cells that secrete cytokinescauses death that influence innate immunity. ApoCells are interactingwith innate immunity, mainly macrophages, monocytes and dendritic cells,to down regulate the response of these macrophages, monocytes anddendritic cells without interacting with T cells or CAR T cells.

Animals that received a high dose of CD19+ CAR T-cells directed to adifferent tumor antigen do not exhibit weight loss or behavioralalterations.

Administration of Apoptotic Cells Inhibits or Reduces the Incidence ofCytokine Release Syndrome Induced by CAR T-Cell Therapy

One group of mice given the highest dose of CD19+ CAR T-cells wasconcomitantly administered 2.10×10⁸/kg apoptotic cells, which waspreviously demonstrated to be a safe and effective dose. Apoptotic cellshad no negative effect in vitro or in vivo on CAR-modified T cells withspecificity against CD19. There were comparable E/T ratios for CART-cells in the presence/absence of apoptotic cells in vitro, andcomparable survival curves in vivo (Data not shown).

Mice receiving human CD19+ CAR T+ apoptotic cells had significantlylowered levels of at least one mouse pro-inflammatory cytokines, lowerweight loss, and reduced mortality.

No negative effect of apoptotic cells on CAR-modified T cells againstCD19 in vivo was seen with comparable E/T ratio results of CAR T in thepresence or absence of apoptotic cells, and a comparable survival curvein vivo.

Significant down regulation (p<0.01) of pro-inflammatory cytokinesincluding, IL-6, IP-10, TNF-α, MIP-1α, MIP-1β was documented (Data notshown). IFN-γ was not downregulated but its effect on macrophages anddendritic cells was inhibited both at the level of phosphorylated STAT1and IFN-γ-induced expression of CXCL10 and CXCL9 (Data not shown.

Conclusion:

CRS evolves from several factors, including tumor biology, interactionwith monocytes/macrophages/dendritic cells, and as a response to the CART cell effect and expansion. Apoptotic cells decrease pro-inflammatorycytokines that originate from innate immunity and inhibit the IFN-geffect on monocyte/macrophages/dendritic cells without harming IFN-γlevels or CAR-T cytotoxicity. Thus, apoptotic cells decreasedpro-inflammatory cytokines that originate from innate immunity andinhibit IFN-γ effect without harming IFN-γ levels and CAR-Tcytotoxicity. These results support the safe use of ApoCells for theprevention of CRS in clinical studies using CAR-T cell therapy.

Example 8: Apoptotic Cells Downregulate Cytokine Release Syndrome (CRS)and Increases CAR-T-Cell Efficacy

Objective:

To test the effects of early apoptotic cells on cytokines and CAR T cellcytotoxicity over an extended time period. To demonstrate the in vivoefficacy of CD19-CAR T-cells. To demonstrate the synergistic effect ofearly apoptotic cells and CD19-CAR T-cells.

Methods:

CD19-expressing HeLa cells (ProMab) were used alone or afterco-incubation with human macrophages for in vitro and intraperitonealexperiments in mice. Raji was used in vivo for leukemia induction. LPSand IFN-g were used to trigger additional cytokine release. Secondgeneration, CD28-bearing, CD19-specific CAR-modified cells were used(either ProMab or produced using a retronectin manufacturing protocol ora polybrene manufacturing protocol) for anti-tumor effect againstCD19-bearing cells. Cytotoxicity assay was examined in vivo (7-AAD flowcytometry) and in vitro (survival curves; tumor load in bone marrow andliver, flow cytometry and immunohistochemistry). CRS occurredspontaneously or in response to LPS and IFN-g. Mouse IL-10, IL-1β, IL-2,IP-10, IL-4, IL-5, IL-6, IFN-a, IL-9, IL-13, IFN-g, IL-12p70, GM-CSF,TNF-a, MIP-1a, MIP-1β, IL-17A, IL-15/IL-15R, IL-7, and 32 humancytokines were evaluated (Luminex technology, MAPIX system; MILLIPLEXAnalyst, Merck Millipore). Mouse IL-6Ra, MIG (CXCL9), and TGF-11 wereevaluated (Quantikine ELISA, R&D systems). IFN-γ effect was evaluated(STAT1 phosphorylation, biological products). Human macrophages anddendritic cells were generated from monocytes. Early apoptotic cellswere produced generally as presented in Example 1 above; ≥40% of cellswere Annexin V-positive; ≤15% were PI-positive.

Mice:

SCID-Bg mice (female, 7-8 wk) were injected with 2 consecutive doses of0.25×10⁶ HeLa-CD19 cells, intra-peritoneally (i.p) on days 1 and 2 ofexperiment. On day 9 mice received i.p. dose of 10×10⁶ 4000-rad (cGy)irradiated ApoCell (using a cell processing system) and an i.p. dose ofa population of 10×10⁶ CAR-T cells comprising either 0.5×10⁶ CAR-Tpositive cells or 2.2×10⁶ CAR-T positive cells, on the following day. Ascontrol, mice received 10×10⁶ activated mononuclear cells or Mock-Tcells. Mice were kept in an SPF animal facility in compliance withinstitutional IACUC guidelines. Mice were weighted twice a week andmonitored daily for clinical signs and peritonitis. End point wasdefined as severe peritonitis manifested as enlarged and tense abdomen,lethargy, reduced mobility or increased respiratory effort. Survivalanalysis was performed according to the Kaplan-Meier method.

Results:

Significant downregulation (p<0.01) of pro-inflammatory cytokines,including IL-6, IP-10, TNF-a, MIP-1a, MIP-1β, was documented (Data notshown). IFN-g was not downregulated, but its effect on macrophages anddendritic cells was inhibited at the level of phosphorylated STAT1 (Datanot shown). IFN-γ induced expression of CXCL10 and CXCL9 in macrophageswas reduced (Data not shown).

In the experiment wherein 0.5×10⁶ CAR-T positive cells were used, 2 miceper group were sacrificed on day 17 and 21. HeLa-CD19 treated miceshowed peritonitis manifested as blood accumulation in the peritoneum,enlarged spleen and tumor loci (Data not shown). Mice treated withcontrol MNCs had a little bit less blood in peritoneum and less tumorloci. Mice treated with CAR-T or with CAR-T and ApoCell had no signs ofperitonitis. This observation correlated to the survival curve presentedin FIG. 20A.

In the experiment wherein 2.2×10⁶ CAR-T positive cells were used, thesame pattern of effect as seen with four-fold fewer CAR T-cells wasobserved (FIG. 20B). CAR-T treatment prolonged survival of mice withperitoneal HeLa-CD19 (p=0.0002). The effect was more significant in thisexperiment, probably due to the higher number of infused CAR T cells(2.2 versus 0.5×10⁶ CAR-T positive cells). Even with the moresignificant and prolonged effect, the early apoptotic cells had asynergistic effect and prolonged survival (p=0.0032). E/T ratios for CART were comparable in the presence/absence of apoptotic cells in vitro.Surprisingly, CAR T cell therapy given in the presence of apoptoticcells ameliorated survival of mice with a significant and reproducibleaddition of at least 12 days (p<0.0032, FIGS. 20A and 20B) in comparisonto CAR therapy alone.

Conclusion:

CAR-T cell treatment prolonged survival of mice with peritonealHeLa-CD19 cells. Administration of irradiated early apoptotic cells oneday before CAR-T had a synergistic effect and prolonged mice survivalfor more than 10 days (p<0.044, log-rank test), compared to CAR-T celltreatment alone.

The irradiated apoptotic cell infusion had a dramatic synergistic effectto CD19-specific CAR T cells in treating CD19-bearing Hela cells in SCIDmice. In this example, similar results were observed to the resultspresented in Examples 5 and 6. By using irradiated apoptotic cells inthis Example, compared with Examples 5 and 6, the possibility of a“graft versus leukemia effect” has been removed. Thus, this surprisingsynergistic effect appears to be mediated via the irradiated apoptoticcells provided.

CRS evolves from several factors, including tumor biology, interactionwith monocytes/macrophages/dendritic cells, and as a response to the CART cell effect and expansion. Apoptotic cells decrease pro-inflammatorycytokines originating from innate immunity, and inhibit the IFN-γ effecton monocytes/macrophages/dendritic cells without harming IFN-γ levels orCAR-T cytotoxicity, and with significant increase in CAR-T cellefficacy. Unexpectedly, treatment with irradiated apoptotic cellscomplements CAR-T cell therapy, effectively extending the anti-cancereffect of the CAR-T cell therapy.

Example 9: Effect of Apoptotic Cells Treatment on a Non-Solid TumorModel

Objective:

To test the effect of apoptotic cells on a non-solid tumor model wherethe cancer is widely spread and not localized or confined, in order todetermine apoptotic cells efficacy on the survival in cancer.

Methods:

Raji Cells

Raji cells were purchased from ECACC (Cat. #: 85011429), and routinelycultured in complete medium (RPMI-1640 supplemented with 10% H.I. FBS,1% Glutamax, 1% Penicillin /Streptomycin), and maintained at aconcentration of 3×10⁵-3×10⁶ cells/ml.

Apoptotic cells were prepared as described in Example 1. Early apoptoticcells produced were at least 50% annexin V-positive and less than 5%PI-positive cells.

Non-Solid (Diffuse) Tumor Model

SCID mice received a single IV injection of 10 Raji cells on day 1 ofthe experiment. A control group of SCID mice received a single IVinjection of saline solution. (3 cohorts were tested; leukemia wasinduced in 2 cohorts using Raji cells, and 1 cohort was maintained as acontrol.)

Solid Tumor Model

SCID mice will receive a single investigational product (IP;Allocetra-OTS) injection of 10 Raji cells on day 1 of the experiment,wherein control groups will receive a single investigational product(IP; Allocetra-OTS) injection of saline solution.

Apoptotic Cell Treatment

In a preliminary study, mice received an infusion of early apoptoticcells 6 days after the infusion of Raji cells. In later studies, themice from one of the leukemic cohorts above received 3 infusions ofearly apoptotic cells (30×10⁶ cells) starting 6 days after the infusionof Raji cells.

Results

SCID mice have no T-cells and therefore no ability to recover fromleukemia without therapy.

Surprising, in the preliminary study and as shown in FIG. 21 , theapoptotic cell infusion (APO) 6 days after the infusion of Raji,significantly prolonged tumor free death in SCID injected with CD19+Raji, compared with mice that did not receive an apoptotic cell infusion(NO APO).

In the leukemic (NO APO) cohort, 70% of mice receiving Raji cellssurvived through their lifespan, compared to 94% of mice receiving bothRaji cells and apoptotic cells (n=51 animals in total, p<0.001). Asexpected, 100% of control mice survived through their expected lifespan(FIG. 22A). In the leukemic cohort, 9% of mice receiving Raji cells andno apoptotic cells survived through up to 12% above the expectedlifespan, compared to 47% of mice receiving both Raji cells andapoptotic cells (FIG. 22B). No mice receiving Raji cells and noapoptotic cells survived through greater than 30% of the expectedlifespan, compared to 41% of mice receiving both Raji cells andapoptotic cells (FIG. 22C). No mice receiving Raji cells and noapoptotic cells attained complete remission, compared to 10% of micereceiving both Raji cells and apoptotic cells (FIG. 22D).

Conclusion:

Administration of an apoptotic cell infusion maintained and increasedthe lifespan of leukemic mice, wherein in certain instances miceadministered early apoptotic cells attained complete remission (FIGS. 2and 3A-3D).

Example 10: Effect of Combined Apoptotic Cell and Anti-CD20 mAbTreatment on a Diffuse Tumor Model

Objective:

To test the effect of administering a combination of early apoptoticcells and anti-CD20 mAb on a diffuse (non-solid) tumor model, whereinthe cancer is widely spread and not localized or confined, in order todetermine the efficacy on survival of this combination therapy.

Raji cells, apoptotic cells, non-solid (diffuse) tumor model, solidtumor model, and apoptotic cell treatment were as described in Examples1 and 9 above.

Anti-CD20 mAb

Commercially available anti-CD20 mAb was acquired from Roche.

Anti-CD20 mAb Treatment

Mice received an IV infusion of 5 mg of anti-CD20 mAb.

Combined Apoptotic Cell and Anti-CD20 mAb Treatment

Starting at day 6 following Raji cell administration, mice receivedthree IV infusions of 30×10⁶ apoptotic cells each. In addition, micereceived an IV infusion of 5 mg of anti-CD20 mAb.

Results

100% of mice receiving Raji cells, Raji cells+anti-CD20 mAb, and Rajicells+antiCD20+apoptotic cells survived through the expected lifespan ofleukemic mice, compared to 86% of mice receiving both Raji cells andapoptotic cells (n=28 animals in total, p<0.0002) (FIG. 23A). No micereceiving Raji cells survived longer than 24% above the expectedlifespan, compared to 29% of mice receiving both Raji cells+apoptoticcells, and 100% of mice receiving either Raji cells+anti-CD20 mAb orRaji cells+antiCD20+apoptotic cells (FIG. 23B). No mice receiving Rajicells survived longer than 59% above the expected lifespan, compared to29% of mice receiving both Raji cells+apoptotic cells, 57% of micereceiving Raji cells+anti-CD20 mAb, and 100% of mice receiving Rajicells+antiCD20+apoptotic cells (FIG. 23C). No mice receiving Raji cellssurvived longer than 76% above the expected lifespan, compared to 29% ofmice receiving both Raji cells+apoptotic cells, 14% of mice receivingRaji cells+anti-CD20 mAb, and 85% of mice receiving Rajicells+antiCD20+apoptotic cells (FIG. 23D). No mice receiving either Rajicells or Raji cells+anti-CD20 mAb survived longer than 100% above theexpected lifespan of a mouse, compared to 29% of mice receiving eitherRaji cells+apoptotic cells or Raji cells+antiCD20+apoptotic cells (FIG.23E).

Conclusion:

Apoptotic cell infusions increased the lifespan of leukemic mice,increased the number of mice attaining complete remission, and enhancedanti-CD20 mAb therapeutic effect (FIGS. 23A-23E).

Example 11: Effect of ApoCell (Early Apoptotic Cells) onLeukemia/Lymphoma

Objective:

The work presented here had three main goals: (1) Evaluating the effectof ApoCell in a leukemia-lymphoma mouse model in terms of disease onset,progression, and ensuing death; (2) Assessing the distribution of tumorcells in a mouse model of leukemia-lymphoma after treatment withApoCell; and (3) Assessing a possible synergistic effect of ApoCell andRituximab (RtX) in the treatment of leukemia-lymphoma in SCID-Bg mice.As part of the work to meet these objectives, measurement of thesurvival of leukemic mice following ApoCell administration was measured.As well, the distribution of tumor cells was measured after treatmentwith ApoCell.

Methods:

Mice.

Female SCID-Bg mice, 7 weeks-old (ENVIGO, Jerusalem, Israel), wereinjected intravenously with 0.1×10⁶ Raji cells per mouse. Mice received3 doses of 30×10⁶ ApoCell intravenously on days 5, 8, and 11 of theexperiment. For combinational therapy, mice received one dose (day 8) ofRtX (2 or 5 mg/kg; Mabthera, Roche, Basel, Switzerland) 1.5 h afterApoCell administration.

Mice were followed daily and weighed twice a week. The endpoint wasdefined as death, or sacrifice due to the development of either of thefollowing symptoms: paraplegia (lower body paralysis), loss of 20% frommouse start weight, lethargy, reduced mobility, or increased respiratoryeffort.

Survival analysis was performed according to the Kaplan-Meier method.Mice were kept in a specific-pathogen-free (SPF) animal facility incompliance with institutional Animal Care and Use Committee (IACUC)guidelines.

Raji Cell Line.

This human Burkitt's lymphoma cell line was purchased from the EuropeanCollection of Authenticated Cell Cultures (ECACC, Cat. #: 85011429), androutinely cultured in complete medium (RPMI-1640 supplemented with 10%heat inactivated FBS, 1% glutamax, 1% penicillin/streptomycin).

Apocell.

Essentially, as described in Example 1. Briefly, an enriched mononuclearcell fraction was collected via leukapheresis from healthy, eligibledonors. Following apheresis completion, cells were washed andresuspended with freezing media composed of PlasmaLyte A pH 7.4, 5%human serum albumin, 10% dimethyl sulfoxide (DMSO), 5% anticoagulantcitrate dextrose solution formula A (ACD-A) and 0.5 U\ml heparin. Cellswere then gradually frozen and transferred to liquid nitrogen forlong-term storage.

For preparation of ApoCell, cryopreserved cells were thawed, washed andresuspended with apoptosis induction media, composed of RPMI 1640supplemented with 2 mM L-glutamine and 10 mM hepes, 10% autologousplasma, 5% ACD-A, 0.5 U\ml heparin sodium and 50 μg/mlmethylprednisolone. Cells were then incubated for 6 hours at 37° C. in5% CO₂. At the end of incubation, cells were collected, washed andresuspended in Hartmann's solution using a cell processing system.ApoCell was centrifuged at 290 g, for 10 min at 2-8° C., and resuspendedin Hartmann's solution for injection. Apoptosis and viability of ApoCellwere determined using Annexin V and propidium iodide (PI, Medical &Biological Laboratories, Nagoya, Japan) using FCS express software.

Flow Cytometry.

Mouse spleen, liver, and bone marrow were collected from sacrificed mice(following deterioration of clinical signs, as defined above) andanalyzed by flow-cytometry (FACSCalibur, BD, Franklin Lakes, N.J., USA)for the presence of the Raji tumor (anti-CD20).

Results:

Part A: ApoCell Delays Disease Onset and Ensuing Death in Leukemic Mice

FIG. 24 is a Kaplan-Meier survival plot presenting 3 individualexperiments (RPMI group, n=15; Raji group, n=23; Raji+ApoCell group,n=24). In each experiment, female SCID-Bg mice (7-8 weeks of age) wereinjected intravenously with 0.1×10⁶ Raji cells and a control group wasinjected with RPMI. Subsequently, mice were administered with threedoses of 30×10⁶ ApoCell by intravenous administration (IV), on days 5,8, and 11. Mice were followed daily and weighed twice a week. Endpointwas defined as death, or sacrifice due to the development of either ofthe following symptoms: paraplegia (lower body paralysis), loss of 20%from mouse start weight, lethargy, reduced mobility, or increasedrespiratory effort. Experimental details are given in Table 11.Significant beneficial effect by ApoCell was seen (p=0.002, Log-rank(Mantel-Cox) test).

TABLE 11 Experimental details of Figure A plot Survival details NumberMean of day of Range Group mice sacrifice (days) Notes RPMI 15 DFS* isshown for all mice Raji 23 22 21-25 Raji + 24 28 19->53 2 mice DFS* onday 53/ Apo Cell 60 (termination of experiment) *DFS = disease freesurvival

The data for the individual studies is presented in FIGS. 25A-25C.

As depicted above, mice treated with 3 doses of ApoCell after theadministration of Raji cells had a slower disease progression, and diedsignificantly later (p=0.0020) than untreated mice.

Leukemic mice treated with ApoCell had a significant delay in the onsetof symptoms, demonstrated a slower disease progression, and died laterthan the control untreated mice. Interestingly, about 10 percent of themice administered with Raji cells and ApoCell did not develop any of theexpected symptoms characteristic in this leukemia/lymphoma model, andremained healthy until termination of the experiments (day 53 or 60).

ApoCell Reduce Tumor Load in Leukemic Mice

Upon sacrifice, following the deterioration of clinical signs asdescribed above, organs of interest were collected for analysis, namelythe liver, spleen, and bone-marrow. Cells of these target organs wereanalyzed by flow cytometry for the presence of human tumor cells (Rajicells are positive for CD20).

The data below (Table 12) describes the average percent cell populationin target organs of the sacrificed mice from the 3 experiments describedabove; values of individual mice in each experiment can be found inTables 13-18, which follow.

TABLE 12 Average percent of tumor population in Spleen, Bone Marrow andLiver (Flow cytometry) # of % CD20⁺ cells Tissue Treatment mice (average± SD) Spleen RPMI 5 0 Raji 10 0 Raji + 8 1.1 ± 3  ApoCell Bone- RPMI 5 0Marrow Raji 10 16.5 ± 8.8 Raji + 8 6.8 ± 9.2 (P = 0.02, ApoCell t-test)Liver RPMI 5 0 Raji 10 11.2 ± 12  Raji + 8 4.5 ± 7.5 (P = 0.06, ApoCellt-test)

In Vivo Experiment 011

FACS Analysis of CD20+ Cells in Bone Marrow, Spleen, and Liver:

One mouse receiving three doses of ApoCell was healthy when sacrificedon day 60.

Liver, spleen, and bone marrow cells were collected and analyzed by flowcytometry (FACSCalibur, BD) for the presence of human CD20-FITC(Biolegend, Cat. #302206); mIgG1-FITC (Biolegend, Cat. #400110).

In Vivo Experiment 019

FACS analysis of tumor cells in bone marrow, spleen, and liver:

Mouse spleen, liver, and bone marrow cells were collected from mice whowere sacrificed following clinical deterioration, as defined in themethods) and analyzed by flow cytometry (FACSCalibur, BD) for thepresence of human CD20 (FITC).

The results of analysis of Spleen, Bone marrow, and Liver are presentedin Tables 13-15 below.

TABLE 13 Spleen Spleen Day of Mouse Treatment sacrifice CD20+ A1 RPMI 0A2 0 A3 0 B2 Raji 22 0 B3 22 0 B5 22 0 B6 22 0 B2 25 0 (020) B3 25 0(020) C1 Raji + 28 0 C2 ApoCell 53 (healthy) 8.7 C4 22 0 C6 22 0 C7 28 0

TABLE 14 Bone Marrow Bone marrow Day of Mouse Treatment sacrifice CD20+A1 RPMI 0 A2 0 A3 0 B2 Raji 22 18.7 B3 22 11.3 B5 22 10.5 B6 22 14.5 B225 21 (020) B3 25 0 (020) C1 Raji + 28 1 C2 ApoCell 53 0.5 (healthy) C422 0.6 C6 22 17.5 C7 28 1.4

TABLE 15 Liver Liver Day of Mouse Treatment sacrifice CD20+ A1 RPMI 0 A20 A3 0 B2 Raji 22 4.4 B3 22 6.5 B5 22 1.7 B6 22 5.4 B2 (020) 25 26.4 B3(020) 25 5.9 C1 Raji + 28 9.8 C2 ApoCell 53 0.5 (healthy) C4 22 3 C6 225 C7 28 22.7

In Vivo Experiment 023

Expression (%) of CD20 tumor cells in the spleen, bone marrow, andliver, as determine by flow cytometry. Mouse spleen, liver, and bonemarrow were collected from sacrificed mice (following clinicaldeterioration, as defined in methods) and analyzed by flow cytometry(FACSCalibur, BD) for the presence of human CD20 (FITC). The results forSpleen, bone marrow, and liver for the individual mice are presented inTables 16-18 below.

TABLE 16 Spleen Spleen Day of Treatment Mouse sacrifice CD20+ Raji B1 220 B2 25 0 B3 22 0 B4 22 0.2 Raji + C1 22 0.2 ApoCell C4 22 0.3 C5 41 0.1C6 47 0 Raji + RtX E1 32 0 2 mg/kg E2 32 0.7 E6 32 0 Raji + RtX F1 40 02 mg/kg + F2 43 0.1 ApoCell F4 35 0 F5 32 0 F6 35 0 F7 57 0.4 Raji + RtXG1 34 0 5 mg/kg G3 34 0.1 G4 43 0 G5 40 0 G7 40 0.1 Raji + RtX H1 40 0.45 mg/kg + H3 36 ApoCell H4 36 H5 40 0 H6 40 0 H7 53 0

TABLE 17 Bone Marrow Bone marrow Day of Treatment Mouse sacrifice CD20+Raji B1 22 18 B2 25 22.5 B3 22 33.6 B4 22 14.9 Raji + C1 22 24.3 ApoCellC4 22 1.8 C5 41 7.8 C6 47 0 Raji + RtX E1 32 0.8 2 mg/kg E2 32 3.1 E6 323 Raji + RtX F1 40 4.1 2mg/kg + F2 43 0.4 ApoCell F4 35 0.2 F5 32 0.8 F635 0.2 F7 57 0.4 Raji + RtX G1 34 2.1 5 mg/kg G3 34 0.5 G4 43 4.3 G5 402.2 G7 40 2.6 Raji + RtX H1 40 0.9 5 mg/kg + H3 36 0 ApoCell H4 36 0 H540 1.6 H6 40 1.3 H7 53 1.8

TABLE 18 Liver Liver Day of Treatment Mouse sacrifice CD20+ Raji B1 226.4 B2 25 42.6 B3 22 5 B4 22 8.1 Raji + C1 22 2.5 ApoCell C4 22 2.2 C541 0.4 C6 47 0 Raji + RtX E1 32 2.1 2 mg/kg E2 32 1.2 E6 32 0.4 Raji +RtX F1 40 0 2 mg/kg + F2 43 ApoCell F4 35 0 F5 32 7.3 F6 35 5 F7 57 0Raji + RtX G1 34 1 5 mg/kg G3 34 7.3 G4 43 1.4 G5 40 0.9 G7 40 5.7Raji + RtX H1 40 0.8 5 mg/kg + H3 36 0.1 ApoCell H4 36 0 H5 40 0.5 H6 4025.1 H7 53 3.4

Preliminary Conclusions:

In conclusion, tumor distribution in the mouse organs correlated thebeneficial effect seen in survival plots and was significantly reducedin bone-marrow and liver in treated mice.

Part B: Synergistic Effect of ApoCell and Rituximab (RtX) in theTreatment of Leukemia/Lymphoma

Next, it was examined whether the ApoCell treatment was synergistic withother conventional treatments of leukemia/lymphoma by evaluating thecombined effect of RtX and ApoCell on leukemic mice in two experiments.

Objectives:

Measurement of the survival of leukemic mice following RtX and ApoCelladministration, and detect tumor cells in bone marrow, liver, and spleenin leukemic mice.

Methods:

The following work is a representative description of the resultsobtained in the combination therapy experiments (ApoCell and rtx).Briefly, female SCID-Beige mice were injected intravenously with 0.1×10⁶Raji cells (n=7 in all groups). Mice received three doses of 30×10⁶ApoCell intravenously on days 5, 8, and 12. On day 8, 1.5 h afterApoCell injection, the mice received a single IV dose of 2 or 5 mg/kgRtX. Mice were followed daily and weighted twice a week. The endpointwas defined as death, or sacrifice due to the development of one or moreof the following symptoms: paraplegia (lower body paralysis), loss of20% from starting weight, lethargy, reduced mobility, or increasedrespiratory effort.

Results:

As shown in FIG. 26 , ApoCell had a beneficial effect corroborating theresults presented in FIG. 24 . Rituxan (RtX) alone had a superior effectto ApoCell both in 2 mg and 5 mg dosage but the combination of ApoCelland Rituxan had a synergistic effect at both in 2 mg (p=0.104) and in 5mg dosage, although the synergistic effect seen in 5 mg did not reachstatistical significance. Tumor distribution in the mouse organscorrelated the beneficial effect (Table 19).

TABLE 19 Statistical analysis of the survival distributions (Log-rank(Mantel-Cox) test) Statistical test (P value) Log-rank Compared groups(Mantel-Cox) Group 1 Group 2 Test Raji Raji + rtx (2 mg/Kg) 0.0002 RajiRaji + rtx (2 mg/Kg) + 0.0002 ApoCell Raji Raji + rtx (5 mg/Kg) 0.0002Raji Raji + rtx (5 mg/Kg) + 0.0002 ApoCell Raji + Rituxan Raji + Rituxan(2 mg/Kg) + 0.0104 (2 mg/Kg) ApoCell

End of Experiment—Day 57

One mouse (Raji+RtX 2 mg/kg+ApoCell) was declared disease free upontermination of the experiment.

The synergistic effect in one 2 mg RtX dose was measured in anadditional experiment. As was clearly shown in this experiment (FIG. 27), the synergistic effect of ApoCell and RtX was again verified assignificant (p=0.01).

As shown in Table 20, the spleen was not populated by tumor cells(0.1-0.3 represents background staining) and was used as a control. Incontrast, bone marrow and liver were tumor targets. There was a reducedtumor population (Rajji-cells, as measured using CD20 marker) in bonemarrow and liver following treatment by ApoCell and RtX separately, andthe benefit increased when the two were given in combination; p=0.0034(**) for Raji+rtx (2 mg/Kg)+ApoCell, and 0.0031 (**) for Raji+rtx (5mg/Kg)+ApoCell (T-test. As expected, RtX significantly reduces tumorburden in the target organs of the leukemic mice. Interestingly,treatment with ApoCell alone reduced tumor cells in those organs tolevels comparable to treatment with conventional RtX therapy.

TABLE 20 Average tumor cell population in the spleen, bone-marrow, andliver (flow cytometry) Statistical # of test (P value) Tissue Treatmentmice CD20+ T-Test Spleen Raji 7 0 Raji +ApoCell 4 0.3 ± 0.4 Raji + rtx(2 mg/Kg) 3 0.2 ± 0.4 Raji + rtx (2 mg/Kg) + 6 0.1 ± 0.1 ApoCell Raji +rtx (5 mg/Kg) 7 0 Raji + rtx (5 mg/Kg) + 4 0.1 ± 0.2 ApoCell Bone Raji 618.3 ± 11   Marrow Raji + ApoCell 4 8.5 ± 11  Raji + rtx (2 mg/Kg) 3 2.3± 1.3 Raji + rtx (2 mg/Kg) + 6   1 ± 1.5 0.0034 ApoCell (**) Raji + rtx(5 mg/Kg) 7 1.7 ± 1.5 Raji + rtx (5 mg/Kg) + 6 0.9 ± 0.8 0.0031 ApoCell(**) Liver Raji 6 15.7 ± 15.4 Raji + ApoCell 4 1.3 ± 1.3 Raji + rtx (2mg/Kg) 3 1.2 ± 0.8 Raji + rtx (2 mg/Kg) + 5 2.5 ± 3.5 ApoCell Raji + rtx(5 mg/Kg) 7   3 ± 2.5 Raji + rtx (5 mg/Kg) + 7 4.4 ± 9.2 ApoCell

Conclusion:

In summary, the survival plots (FIG. 24 , FIG. 26 , and FIG. 27 )clearly demonstrate the beneficial effects of ApoCell and Rtx along aswell as the synergistic effect of ApoCell and RtX in combination. Ofnote, when the conventional RtX treatment was combined with ApoCell,survival times increased significantly, regardless of the RtX dose,indicating a potent synergistic effect of the two therapies. Asupporting clinical observation was the decrease in the of tumor cellpopulation in the bone marrow and liver (Table 12).

Surprisingly, ApoCell preparation had a remarkably beneficial effect ondisease progression and survival in a leukemic mouse model, independentof any other treatment. 10-20% of mice had prolonged survival inKaplan-Meier analysis (FIG. 24 , FIG. 26 , and FIG. 27 ), and the tumorcell burden was reduced in the liver and bone marrow. Furthermore, therewas a marked synergistic effect when ApoCell and RtX were administeredin combination, further delaying disease onset and progression, andimproving survival.

Example 12: Use of Pooled Apoptotic Cell Preparation in GVHDLeukemia/Lymphoma Models

In the following preliminary work, the effect of the same infusion inGvHD leukemia/lymphoma models was examined. The safety and efficacy ofan irradiated multiple donor single apoptotic cell infusion (a pooledmononuclear irradiated apoptotic cell preparation) for the prevention ofacute GvHD in mice undergoing bone marrow transplantation (BMT) wasexamined. In this model, BMT rescued irradiated mice (80-100%).

The question regarding the possible loss of graft versus leukemia (GvL)effect arises in every successful treatment that potentially avoids highgrade aGVHD, since this effect was found to correlate with the severityof GVHD.

Methods

Apoptotic cells were prepared as per Example 1 above, except that in thecurrent experiments, preparation was done simultaneously from 4 donors.Following preparation from 4 donors, the cell preparations were combinedat the last step (prior to irradiation), irradiated immediately after,and injected immediately after irradiation. Irradiation was at 25 Gy.

Results

The two graphs presented in FIGS. 28 and 29 , show the clear effect(p<0.01) of a single injection of apoptotic cell from multipleindividual donors (dotted line), both on survival and weight loss. FIG.28 is a Kaplan-Meier survival curve in a GvHD mouse model that wastreated with a single dose irradiated apoptotic cells from multipleindividual donors where survival was significantly ameliorated. FIG. 29is percentage of weight loss of the 2 compared groups that follow andcorrelate with the findings of FIG. 28 .

In summary, the single infusion of multiple-donor irradiated apoptoticcells successfully and significantly improved life expectancy in a mousemodel of GvHD.

Example 13: Stability Criteria for Apoptotic Cells from MultipleIndividual Donors

The objective of this study is to develop stability criteria forapoptotic cells from multiple individual donors with comparabilitystudies to non-irradiated HLA-matched apoptotic cells (Mevorach et al.(2014) Biology of Blood and Marrow Transplantation 20(1): 58-65;Mevorach et al. (2015) Biology of Blood and Marrow Transplantation21(2): S339-S340).

Apoptotic cell final product preparations will be evaluated for cellnumber, viability, apoptotic phenotype and potency after storage at 2 to8° C. for 8, 24, 48, and 60 hours with sampling at each time point.Apoptotic cell final product lots will be prepared following standardoperating procedures (SOPs) (Example 1; Example 5) and batch records(BRs; i.e., specific manufacturing procedures). For potency evaluation,samples of early apoptotic cell preparation final product lots will betested for inhibition of lipopolysaccharide (LPS) induced upregulationof MHC-II expression on immature dendritic cells (time points 0-24 h) ormonocytes (time points 0-6) and will be performed according to SOPs andrecorded on BR. These series of test will be performed on pooled andnon-pooled products that are in preparations originating from multipleindividual donors and from single donors, respectively.

In addition, flow cytometric analysis of CD3 (T cells), CD19 (B cells),CD14 (monocytes), CD15^(high) (granulocytes) and CD56 (NK cells) will bedocumented. The aims of these studies are to demonstrate consistencywith a narrow range of results. Preliminary results are consistent withthese goals and no deviations from the SOP are noted and no technicalproblems are reported. However, further studies are needed in order toconclude the range and stability of effective treatment. Preliminaryresults show equivalence in all these parameters. Further, single donorstability studies showed stability at least through a 48 hour period(See, Example 1).

Example 14: Safety & Efficacy of Multiple Donor Irradiated ApoptoticCells as Prophylaxis For Acute Graft-Versus-Host Disease

Objective:

A phase 1/2a, multicenter, open-label study evaluating the safety,tolerability and preliminary efficacy of a single dose administration ofirradiated apoptotic cells, from multi-, unmatched-donors, for theprevention of graft versus host disease in hematopoietic malignancies inhuman leukocyte antigen-matched, related and unrelated patientsundergoing allogeneic h1a-matched hematopoietic stem celltransplantation

Primary Objective:

To determine safety and tolerability of multiple donor irradiatedapoptotic cell treatment.

Secondary Objective:

To determine efficacy of irradiated apoptotic cells from multipleindividual donors as prophylaxis measure for acute GVHD (aGVHD) inpatients with hematopoietic malignancies scheduled to undergohematopoietic stem cell transplantation (HSCT). For the purposes of thisstudy, HSCT can be either bone marrow transplant (BMT) or peripheralblood stem cell transplantation (PBSCT).

Therapeutic Indication:

Graft vs. Host Disease (GVHD) post-transplantation in hematopoieticmalignancies in human leukocyte antigen (HLA)-matched, related andunrelated patients

Study Design:

This is an open labeled study, multi-center, phase-1/2a study inpatients diagnosed with hematopoietic malignancies scheduled to undergoHSCT (either bone marrow transplantation or peripheral blood stem celltransplantation) from an HLA-matched related or unrelated donor,following either full myeloablative or reduced intensity myeloablativeconditioning regimens.

After a signing of informed consent by recipient patient, donorsscreening period and cell collection before initiating conditioningregimen, eligible recipient patients will be assigned (stratified byprophylactic treatment and related versus non-related transplant donorsin 1:1 ratio to receive intravenous (IV) injection 12-36 hours prior toHSCT transplantation to either:

Investigational Arm:

single dose of 140×10⁶±20% cell/kg from multiple individual donors ofirradiated early apoptotic cells/kg body weight in phosphate buffersolution (PBS).

All patients will also be treated with the institutional standard ofcare (SOC) immunosuppressive regimen: cyclosporine/methotrexate ortacrolimus/methotrexate for full myeloablation andmycofenolate/cyclosporine or mycophenolate/tacrolinus for reducedintensity. Patients will be hospitalized as medically indicated.

Patients will be followed up for 180 days for the secondary efficacyendpoint and for 1 year for the primary safety and tertiary efficacyendpoints. Number of visits for patients participating in this studywill be comparable to those customary for patients in their condition.For donor, study specific visit will be for apheresis procedure duringthe screening period.

As these patients have many underlying medical conditions and mayexperience symptoms compatible with aGVHD, it may be difficult toabsolutely determine if toxicity is related to apoptotic cells or notalthough basic data exist from a former phase 1-2a study using apoptoticcells for GvHD prophylaxis (Mevorach et al. (2014) Biology of Blood andMarrow Transplantation 20(1): 58-65) Single Infusion of DonorMononuclear Early Apoptotic Cells as Prophylaxis for Graft-versus-HostDisease in Myeloablative HLA-Matched Allogeneic Bone MarrowTransplantation: A Phase I/IIa Clinical Trial. BBMT 20(1)58-65).

Data Safety Monitoring Board (DSMB) will meet as specified in the DSMBcharter, including at the time of the scheduled interim analysis (180days) assuming no safety concerns were raised beforehand.

Study Procedures:

The study will comprise of screening, treatment and follow-up periods.

1. Screening Period (Day −60 to Day −2)

Potential recipient patients will sign informed consent prior to conductof any study related procedures. The standard assessments beforeapproval, will be performed by the transplantation center for the donorduring the screening period and usually include: demographic data,medical history, HLA match status verification (no matching is needed),physical examination, height and weight, vital signs, pregnancy test(all women), hematology, blood chemistry, infectious disease screen, ECGand urinalysis.

The recipients (study patients) will undergo the following assessmentsduring the screening period: demographic data, medical history,Karnofsky performance status, HLA match verification, physical exam,height and weight, vital signs, pregnancy test (all women), ECG,pulmonary function test, hematology, blood chemistry, coagulationmarkers, infectious disease screen, and urinalysis.

After the initial screening evaluations, if recipient is eligible toparticipate in the study, the recipient patient will be assigned on thefirst day of the conditioning regimen to receive single IV infusion of140×10⁶±20% cell/kg of multiple donor apoptotic cells. The conditioningregimen to be completed on the day before or day of Apoptotic Cellinfusion scheduled for Study Day −1.

Apoptotic cell dosage will be calculated for each recipient patient andpresumed apheresis collection number and number of donors will bedecided accordingly.

For Peripheral Stem Cell Transplant Donors:

Between Days −6 to −1, the donor will receive one or more once dailyinjections of G-CSF to mobilize progenitor cells and on Day 0 willundergo apheresis to produce donor hematopoietic blood stem cells fortransplantation. Preparation of the hematopoietic blood stem cells forbone marrow transplantation will be performed in accordance with thecenter's standard practice by trained hospital staff. The hematopoieticblood stem cells for HSCT will not be manipulated or T cell-depletedprior to administration.

For Bone Marrow Transplant Donors:

Bone marrow will harvested and prepared per center standard practice andwill not be otherwise manipulated.

2. Treatment Day (Day −1)

On Day −1 (12-36 hours prior to HSCT), eligible patients will receivesingle IV infusion of either 140×10⁶±20% cell/kg of multiple individualdonors irradiated Early apoptotic Cellsl. Vital signs will be monitoredevery hour during infusion and every 4 hours for the first 24 hoursafterwards. Treatment-related AEs will be assessed immediately followinginfusion.

On Day 0, patients will undergo hematopoietic stem cell transplantationaccording to local institution guidelines.

3. Short-Term Follow-Up Period (Day 0 to Day 180)

Patients will be followed-up to Study Day 180 for assessment of theprimary endpoint safety and tolerability and secondary and tertiaryendpoints: cumulative incidence of aGVHD grade II-IV (“modifiedGlucksberg” consensus based on Przepiorka et al cumulative incidence ofany grade and high grade aGVHD, i.e., time to development of aGVHD,grades II-IV; any systemic treatment of GVHD, and the development ofcGVHD.

The short term follow up visits will be daily while hospitalized for thetransplantation (usually at least Days −1 to +14 or more) and weeklyvisits during the first 7 weeks after discharge; days +7, +14, +21. +28,+35, +42, and then on Days 60, 100, 140, and 180. The visit window willbe ±5 days for each weekly visit (first 7 weeks) and ±5 days forbiweekly or more visits during the subsequent follow up period up to 180days.

Blood samples will be obtained on days 1, 3, 7, +7, +28, +42, 60, 100,140 and 180 and examined for documentation of engraftment, immunologicalrecovery, plasma and serum biomarkers (“Michigan”) and cellsubpopulations.

4. Long-Term Follow Up Period (Day 181 to Day 365/1 Year)

Patients will be followed for one year post-HSCT for the longer termsecondary endpoints: non-relapse mortality and overall survival (OS),relapse incidence, leukemia free survival (LFS) and chronic GVHD. Therewill be at least two long-term follow-up visits, the last one being,12±1 months following the HSCT.

Study Duration:

For each participating patient, the duration in the study will be up to14

months as follows:

Screening Up to 60 days (2 months

Treatment 1 day

Follow-up 365 days (12 months) consisting of

Short-term: 180 days

Long-term +180 days

Study Population:

A total of 25 patients diagnosed with hematologic malignancies scheduledto undergo HSCT (either bone marrow transplantation or peripheral bloodstem cell transplantation), with at least 15 unrelated donors, followingeither myeloablative or reduced intensity conditioning regimens, percenter standard practice will be included in this study and will becompared to historical controls.

Inclusion/Exclusion Criteria:

Recipient Patient Exclusion Criteria

1. Patients, Age >18, who are eligible for allogeneic HSCT for thefollowing malignancies:

-   -   Acute myeloid or undifferentiated or biphenotypic, leukemia, in        complete remission (any remission) or beyond but with <5% blasts        by morphology in bone marrow.    -   Acute myeloid leukemia (AML) in complete remission if it has        evolved from myelodysplastic syndrome (MDS) (there should be        documented diagnosis of MDS at least 3 months prior to diagnosis        of acute myeloid leukemia). Or evolved from polycythemia vera or        essential thrombocytosis.    -   Acute lymphoblastic leukemia (ALL) in complete remission (any        remission) with <5% blasts by morphology in bone marrow.    -   Chronic myeloid leukemia (CML) in chronic or accelerated phase    -   Myelodysplastic syndromes—refractory cytopenia with multilineage        dysplasia (RCMD), RA (refractory anemia), RA with ringed        sideroblast (RARS; all <5% blasts), RA with excess blasts (RAEB;        5 to 20% blasts).

The transplant donor and recipient patient must have at least an 8/8 HLAmatch at the HLA A, B, C, DQ, and DR loci and no antigen or allelemismatch. However the donor(s) of leukocytes for apoptotic cellformation is not restricted to HLA matching.

Performance status score of at least 70% at time of the screening visit(Karnofsky for adults and Lansky for recipient <16 years old.

Cardiac left ventricular ejection fraction ≥40% in adults within 4 weeksof initiation of conditioning; MUGA scan or cardiac ECHO required ifprior anthracycline exposure or history of cardiac disease.

Pulmonary function test with DLCO¹, FEV1 (forced expiratory volume) andFVC (forced vital capacity) of ≥60% predicted. Diffusing capacity of thelung for carbon monoxide

Oxygen saturation of at least 90% on room air.

Patients must have adequate organ function as defined below:

AST (SGOT)/ALT (SGPT)<3×upper limit of normal (ULN).

Serum creatinine <2.0 mg/dL (adults, >16 y) or <O.8 (1-2 y), <1(3-4 y),<1.2 (5-9 y), <1.6 (10-13 y), and 1.8 (14-15 y).

Serum bilirubin <3 mg/dL unless due to Gilbert's disease or hemolysis.

Signed written informed consent to participate in the studyindependently by patient, or guardian in the case of minors.

Ability to comply with the requirements of the study.

For duration of 4 weeks (from day −1), both female and male must agreeto: Use an acceptable method of birth control or be surgically sterilefor the first month or more if there are BMT related restrictions.

To have a negative pregnancy test regardless of child-bearing potential.

Recipient Patient Exclusion Criteria

All diseases eligible for HSCT not specified in the Inclusion Criteria.

Participation in an interventional investigational trial within 30 daysof the screening visit.

Have progressive or poorly controlled malignancies.

If BMT plan include T-cell depleted allograft

If BMT plan include anti-thymocyte globulin (ATG) or alemtuzumab as partof immunosuppressive regimen or high dose Cyclophosphamide therapy forthe prevention of GVHD after transplantation

Uncontrolled infections including sepsis, pneumonia with hypoxemia,persistent bacteremia, or meningitis within two weeks of the screeningvisit.

Current known active acute or chronic infection with HBV or HCV.

Known human immunodeficiency virus (HIV) infection.

Patients with severe or symptomatic restrictive or obstructive lungdisease or respiratory failure requiring ventilator support.

Patients with other concurrent severe and/or uncontrolled medicalcondition which could compromise participation in the study (i.e. activeinfection, uncontrolled diabetes, uncontrolled hypertension, congestivecardiac failure, unstable angina, ventricular arrhythmias, activeischemic heart disease, myocardial infarction within six months, chronicliver or renal disease, active upper gastrointestinal tract ulceration).

Any chronic or acute condition susceptible of interfering with theevaluation of investigational product effect.

Any form of substance abuse (including drug or alcohol abuse),psychiatric disorder or any chronic condition susceptible, in theopinion of the investigator, of interfering with the conduct of thestudy.

Organ allograft or previous history of stem cell transplantation(allogeneic only).

Breast feeding in women of childbearing potential.

Patients who are likely to be non-compliant or uncooperative during thestudy.

Investigational Product Route and Dosage Form

Apoptotic cells will be administered as an IV infusion of 140×10⁶+20%cell/kg of irradiated multiple donor apoptotic cell product 12-36 hoursprior to HSCT.

Apoptotic cells are a cell-based therapeutic composed of multipleindividual donors apoptotic cells. The product contains allogeneic donormononuclear enriched cells in the form of liquid suspension with atleast 40% early apoptotic cells. The suspension is prepared frommultiple individual donors with PBS solution in accordance with GMPregulations and should be stored at 2-8° C. until infusion. The finalproduct will be in a total volume of 300-600 mL in an opaque transferpack and will be irradiated with 25 Gy following preparation.Investigational product should be administered to the patient within 48hours of completing the manufacturing process.

Safety Outcomes/Efficacy Endpoints/Outcome Measures

Primary:

Safety and tolerability endpoints include time to engraftment and aphysical examination to determine adverse events, concomitantmedications and safety laboratories on Day 180 and Day 360 (1 year).Further, it is expected that irradiated pooled apoptotic cellpreparations will show a lack of in vitro and in vivo cell proliferationand lack of in vivo activation. Such a showing identifies the pooledapoptotic cell preparation as safe for use.

Secondary:

Cumulative incidence of aGVHD grade II-IV using “modified Glucksberg”consensus based on (Rowlings et al. 1997) IBMTR Severity Index forgrading acute graft-versus-host disease: retrospective comparison withGlucksberg grade. Br J Haematol. 1997 June; 97(4):855-64) on Day 1801-year non-relapse mortality and overall survival (OS)1-year relapse incidence1-year leukaemia free survival (LFS)Maximum grade of aGVHD within the first 180 daysCumulative incidence of grade III-IV aGVHDIncidence of chronic GVHD according to (Jagasia et al., 2015) on Days180 and 360 (1 year).Any “systemic treatment” including corticosteroids (both used or not andcumulative dosage) for the treatment of aGvHD on Day 20 through Day 180Immune reconstitution and function on Days +28, 100, 180 and 360 (1year) in relation to T, B, NK, and MonocytesMajor infection rate (including lung infiltrates, CMV reactivation andany other infections that require hospitalization) through Day 180 and 1year.

Tertiary/Exploratory:

Percent of hospitalization days to total days at risk, or total daysalive and out of the hospital. Or total hospitalization days till firstdischarge post transplantation.

Organ specific GVHD

T regs, CD4 Tcon, CD8, NK and B cells levels on Day 180

Statistical Analysis:

Study outcome will be compared to historical control with individualswith comparable baseline characteristics.

Descriptive statistics will be used to summarize outcome measures andbaseline characteristics. In this analysis all available data will bepresented with no imputation for any missing data. Subjects willcontribute the data available up to the point of withdrawal or studycompletion or death. The descriptive statistics such as means, median,standard deviation, minimum and maximum will be used to summarizecontinuous variables. All subjects who receive the apoptotic cellsinfusion will be included in the safety analysis. Subjects who alsoreceive the HSCT will be included in the efficacy analysis. As thisstudy is exploratory in nature, ad hoc analyses are planned.

Sample Size Consideration

A total of 25 patients will be included at least 15 matched unrelatedpatients will be enrolled. Apoptotic cells (active will be given to all,stratifying on GVHD prophylaxis regimen, and related versus unrelatedtransplant donor.

Population Analysis Definition

All efficacy analyses will be conducted on the Intent-to-Treat (ITT)population and compared to adequate historical control. The safetypopulation will be defined as all patients who receive a dose of studymedication.

Statistical Methods

Patient, disease, and transplant characteristics will be described usingfrequencies and percentages or median (range) as appropriate.

Safety Analysis

Descriptive statistics will be used to summarize safety outcomes withfocus on the AEs reported between study treatment infusion and HSCTprocedure (24-30 hour window). No alterations in the conduct of thestudy will be initiated as a consequence of the DSMB review, includingsample size adjustment. As such, no penalty adjustment in the overallType I error as a consequence of the interim analysis will be required.

Secondary Endpoint Analysis

Grade II-IV aGVHD will be described using the cumulative incidenceestimator with death prior to aGVHD as a competing event.

Neutrophil and platelet recovery, Grade III-IV aGVHD, chronic GVHD,infection, relapse, and transplant related mortality will be describedusing cumulative incidence with relapse as competing event for TRM anddeath as the competing event for all others. Overall survival andleukemia free survival will be described using the Kaplan-Meierestimator, and. The maximum grade of aGVHD within the first 180 days andthe need for steroids at 180 days will be described using frequenciesand percentages using the Mann-Whitney U-test and chi-square testrespectively. Immune recovery of each cell subset and TREGs will bedescribed at each time point using median and range Mann-Whitney tests.

Example 15: Comparison of Pooled Apoptotic Cell Preparation Vs. SingleDonor Apoptotic Cell Preparation in GVHD Leukemia/Lymphoma Models

Objective:

Compare the beneficial clinical effect of human early apoptotic cellsobtained from a single donor on the severity of GvHD in a murine modelof GvHD, to the clinical effect, if any, of human early apoptotic cellsobtained from multiple individual donors on the severity of GvHD in themurine model of GvHD, wherein the multiple individual donors representedHLA-unmatched heterologous donors.

Example 12 above shows the beneficial effect of irradiated apoptoticcells pooled from multiple individual donors. The results shown in FIG.28 and FIG. 29 were surprising as a skilled artisan may recognize thatthe multiple sources of unmatched cells may have increased the diversityof antigenicity of the cells, and thus would have expected a dramaticreduction in the clinical effect. Unexpectedly, the known, beneficialeffect of early apoptotic cells on the reduction of GvHD severity, andtherefore a prolongation of the number of days till mortality, was alsoalleviated by pooled unmatched early apoptotic cells (FIG. 28 ), whichwould purportedly have increased antigenicity due to the pooled multipleunmatched source cells.

An additional objective was to understand if there is a differencebetween the use of irradiated early apoptotic cells and non-irradiatedapoptotic cells.

A skilled artisan would appreciate that unmatched, irradiated cells keeptheir antigenic profile as recognized by the APC mechanism and so byT-Cells of the host into which they have been infused. Accordingly,concerns when pooling heterologous unmatched populations of cellsincluded cross-reactivity between the individual populations beingpooled, mixed-cell lymphatic reactions of pooled populations, or T-cellimmune reactions between pooled populations that could reduce oreliminate cells, or any combination thereof.

Methods

Mouse Model:

Female 7-9 week-old BALB/c mice (H-2^(d)) were used as recipients andfemale 8-9 week-old C57BL/6 mice (H-2^(b)) were used as donors inmismatched GVHD model. Recipients were total body irradiated at 850 cGy24 hours before bone marrow and splenocyte transplantation. Donorbone-marrow cells were used for bone-marrow reconstitution. Bone marrowcells were extracted from the femoral and tibial bones with RPMI 1640.Red blood cells were lysed, then cells were washed and resuspended withPBS. Viability was assessed using trypan blue dye exclusion (>90%viability). Donor splenocytes were used for the induction of GVHD.Spleens were removed and homogenized and single cell suspension wasobtained. Red blood cells were lysed and splenocytes were resuspendedwith PBS. At least 90% viable cells were assessed using trypan blue dye.

Early apoptotic cells: Apoptotic cells were produced from mononuclearenriched cell fraction apheresis from healthy donors similar toExample 1. In brief:

Enriched fractions of mononuclear cells (MNCs) were obtained fromhealthy, eligible donors via leukapheresis procedure. Cells werecollected via Spectra OPTIA® apheresis system from 12 liters of blood,in addition to 400-600 ml of autologous plasma. The estimated yield ofthe enriched mononuclear cell fraction from a donor was expected to beapproximately 1.2-1.5×10¹⁰ cells. Prior to leukapheresis procedure,donors are tested and confirmed negative to the below viral vectors:

-   -   1. Human Immunodeficiency virus (HIV), types 1 and 2;    -   2. Hepatitis B virus (HBV);    -   3. Hepatitis C virus (HCV);    -   4. Cytomegalovirus (CMV);    -   5. Treponema pallidum (syphilis);    -   6. Human T-lymphotropic virus (HTLV), types I and II

Following cell collection, the cells were washed with RPMI and frozen asfollows. The freezing formulation was composed of PlasmaLyte A forinjection pH 7.4, 10% DMSO, 5% Human Serum Albumin and 5% AnticoagulantCitrate Dextrose solution inoculated with 10 U\ml heparin.

Freezing media was prepared in bags and the freezing procedure performedin a closed system under cGMP conditions.

Following leukapheresis procedure completion, enriched MNC fraction waswashed with PlasmaLyte A and resuspended with ice-cold freezing media toa concentration of 50-65×10⁶ cells\ml. Cells were then transferred tofreezing bags, bags were transferred to pre-cooled aluminum cassettesand cassettes were transferred immediately to −18-(−25°) C for twohours.

Following the two hours, cassettes were transferred to −80° C. for anadditional 2 hours and then to long-term storage in liquid nitrogen(>−135° C.).

Autologous plasma was divided to 50 gr aliquots. Plasma aliquots weretransferred to −80° C. for 2 hours and then to a long-term storage in−18-(−25)° C.

For apoptosis induction cells were thawed and washed with pre-warmedRPMI1640 containing 10 mM Hepes buffer, 2 mM L-Glutamine and 5%Anticoagulant Citrate Dextrose solution inoculated with 10 U\ml heparin.After supernatant extraction cells were resuspended at finalconcentration of 5×10⁶/ml in RPMI 1640 supplemented with 10 mM Hepes, 2mM L-glutamine, with addition of 10% autologous plasma and. 50 μg\mlMethylprednisolone and 5% Anticoagulant Citrate Dextrose solutioninoculated with 10 U\ml heparin. Cells are then transferred to cellculture bags, and incubated at humidified incubator 37° C., 5% CO₂ for 6hours to stabilize apoptosis.

Following incubation cells were harvested, washed with PBS andresuspended in PBS.

Early apoptotic cell product was produced from one single donor orcombined 10 different individual donors, in which case cells werecombined just prior to irradiation. Since interference may occur betweencomponents in the multiple donor product, for example between livingnon-apoptotic cells, the early apoptotic cell product was subdivided anda sample of early apoptotic cells to be tested in vivo was irradiatedwith 2500 cGy prior to administration (sample F below), and stored at2-8° C. until administration. Table 3 of Example 6 below presentsdetails of the Annexin V positive/Propidium iodide negative ratio andcell surface markers of the early apoptotic cell product, establishingthat consistency of apoptotic cells administered to mice is maintained.The final product was stable for 48 hours at 2-8° C.

On the day of transplantation, mice received 5×10⁶ bone-marrow cells,3×10⁶ splenocytes and 30×10⁶ single- or multiple-donor early apoptoticcell product, according to the following experimental design:

Irradiation Control

Reconstitution control—irradiation+Bone-Marrow transplantation (BM)

GVHD control—irradiation+Bone-Marrow and splenocyte transplantation

Single donor, irradiated—irradiation+Bone-Marrow and splenocytetransplantation+irradiated early apoptotic cell product from singledonor

Single donor, non-irradiated—irradiation+Bone-Marrow and splenocytetransplantation+non-irradiated early apoptotic cell product from singledonor

Multiple donor, irradiated—irradiation+Bone-Marrow and splenocytetransplantation+irradiated early apoptotic cell product from multipledonor

Multiple donor, non-irradiated—irradiation+Bone-Marrow and splenocytetransplantation+non-irradiated early apoptotic cell product frommultiple individual donors.

Monitoring—Transplanted mice were tagged and survival was monitored.Body weight was assessed every two days for the first two weeks of theexperiment and then every day. Loss of 35% from initial body weight wasdetermined as primary end point and mice were sacrificed and survivalcurve was updated accordingly. Body weight results were comparable tothose observed in Example 12 FIG. 29 .

The severity of GVHD was assessed using a known scoring system (Cooke KR, et al. An experimental model of idiopathic pneumonia syndrome afterbone marrow transplantation. I. The roles of minor H antigens andendotoxin. Blood. 1996; 8:3230-3239) that incorporates five clinicalparameters: weight loss, posture (hunching), activity, fur texture andskin integrity. Mice were evaluated and graded from 0 to 2 for eachcriterion. By summation of the five clinical scores a clinical indexvalue was generated (index number increases with the severity of GVHD).

Results

Percent survival of the different population of mice is presentedgraphically in FIG. 30 . The irradiation only control mice died betweenday 8 and 12 (n=13), as expected from mice that did not receive bonemarrow reconstitution. The majority of GVHD control mice (receivedbone-marrow and spleen) died between day 6 and 27. One mouse did not die(n=18). In bone-marrow reconstitution control group (BM) 3 out of 7 micedied between day 6 and 8. In the remaining mice, bone marrow wasreconstituted by donor bone-marrow and mice remained alive (>50 days).

Single donor, non-irradiated mice died between day 15 and 36. Thus, aspreviously shown, single donor non-irradiated early apoptotic cells hada beneficial effect and survival was prolonged (p<0.01).

Single donor, irradiated mice died between day 7 and 35, one mouseremained disease free survival (>50 days). This demonstrated that singledonor irradiated apoptotic cells also provided the beneficial effectwith respect to GVHD. Thus, irradiation did not harm theimmunomodulatory effect of early apoptotic cells. All had beneficialeffect on survival in the GVHD murine model compared to GVHD control(p<0.01).

Non-irradiated multiple donor treatment did not provide a beneficialeffect compared to GVHD control (n=1). Survival pattern was similar toGVHD control and mice died between day 6 and 28 (p=NS—not significant).Surprisingly and in contrast to the non-irradiated apoptotic cells,irradiated-multiple individual donor apoptotic cells (treatment F)(n=10) had a beneficial effect similar to single donor treatment, ascompared with GVHD control. GVHD symptoms appeared significantly laterand mice died between day 18 and 34 (p<0.01).

Irradiated-multiple individual donor (n=10), irradiated single donor(n=10) and non-irradiated single donor treatment (n=10) had similarsurvival patterns and no significant difference in effect on survivalwas observed between these three treatment groups.

The experiments indicated a clear effect of apoptotic cells infusion inGVHD induced mice. There was a significant prolonged survival effect forthe treatments of irradiated multiple individual donors and irradiated-and non-irradiated single donor apoptotic cells.

Multiple donor treatment did not prolonged survival of mice when notirradiated but the irradiation of the apoptotic cell product prior toadministration to mice improved results and treatment had close survivalpattern as single-donor treatments.

As stated above, FIG. 30 shows, non-irradiated apoptotic cells obtainedfrom multiple unmatched donors have significantly lower positiveclinical effect on reduction in GvHD and mortality (% survival), ascompared to (1) non-irradiated apoptotic cells obtained from singleunmatched donors; (2) irradiated apoptotic cells obtained from singleunmatched donors; and (3) irradiated apoptotic cells obtained frommultiple unmatched donors. In addition, all three (non-irradiated earlyapoptotic cells, single donor; irradiated early apoptotic cells, singledonor; and irradiated early apoptotic cells, multiple individual donors)have similar effects.

This data was surprising since the antigenicity of the non-irradiatedapoptotic cells obtained from multiple individual donors was expected tobe similar to that of irradiated apoptotic cells obtained from multipleindividual donors, why would not both have similar hostile antigenicreaction with the implanted bone marrow, and why would both not be ableto reduce GvHD and mortality rate?

If antigenicity is the main issue here, it was expected to seedifferences between the clinical effects of non-irradiated apoptoticcells obtained from single donor and irradiated apoptotic cells obtainedfrom single donor. However the data does not show this difference.

One possibility is that the lack of efficacy of non-irradiated pooledapoptotic cell preparations prepared from multiple individual donors,resulted from cross-interaction between the individual mononuclearpopulations present in the pooled preparation. These interactions do notappear to be directly attributable to antigenicity towards the host, asirradiated cells maintain their antigenicity but the efficacy differedsignificantly from non-irradiated cells. Therefore, it appears that thecross-interaction in the pooled early apoptotic cell preparationsreceiving irradiation was unexpectedly eliminated and the host respondedwell to administration of the cells.

As shown, irradiated pooled donors had essentially the same effect as asingle non irradiated donor.

Example 16: EFFECT OF IRRADIATION ON FINAL APOPTOTIC CELL PRODUCT

Apoptotic cells are increasingly used in novel therapeutic strategiesbecause of their intrinsic immunomodulatory and anti-inflammatoryproperties. Early apoptotic cell preparations may contain as much as20-40% viable cells (as measured by lack of PS exposure and no PIadmission; Annexin V negative and Propidium iodide negative) of whichsome may be rendered apoptotic after use in a transfusion but some willremain viable. In the case of bone marrow transplantation from a matcheddonor, the viable cells do not represent a clinical issue as therecipient is already receiving many more viable cells in the actualtransplant. However, in the case of a third party transfusion, (orfourth party or more as may be represented in a pooled mononuclearapoptotic cell preparation) use of an apoptotic cell population thatincludes viable cells may introduce a second GvHD inducer. Furthermore,the implication of irradiation on the immunomodulatory potential ofearly apoptotic cells has so far been not assessed. A skilled artisanmay consider that additional irradiation of an early apoptotic cellpopulation may lead cells to progress into later stages of apoptosis ornecrosis. As this appears a particularly relevant question with regardto clinical applications, the experiments presented below were designedto address this issue, with at least one goal being to improve thebiosafety of functional apoptotic cells.

Thus, the aim was to facilitate the clinical utilization of apoptoticcells for many indications wherein the potency of apoptotic cells mayrely on a bystander effect rather than engraftment of the transplantedcells.

Objective:

Examine the effect of irradiation on early apoptotic cells, whereinirradiation occurs following induction of apoptosis.

Methods (in Brief):

The cells were collected according and early apoptotic cells wereprepared essentially as described in Example 5.

Three separate early apoptotic cell batches were prepared on differentdates (collections 404-1, 0044-1 and 0043-1).

Each final product was divided into three groups:

Untreated

2500 rad

4000 rad.

Following irradiation, early apoptotic cells were tested immediately(to) for cell count, AnnexinV positive-PI negative staining, cellsurface markers (% population of different cell types) and potency(dendritic cells (DCs)). Following examination at to, early apoptoticcells were stored at 2-8° C. for 24 hours, and examined the next dayusing the same test panel (t_(24h)) (cell count, Annexin V positive-PInegative staining, and cell surface markers and potency).

Previously, a post-release potency assay was developed, which assessesthe ability of donor mononuclear early apoptotic cells (Early apoptoticCellsl) to induce tolerance (Mevorach, D., T. Zuckerman, I. Reiner, A.Shimoni, S. Samuel, A. Nagler, J. M. Rowe, and R. Or. 2014. Singleinfusion of donor mononuclear early apoptotic cells as prophylaxis forgraft-versus-host disease in myeloablative HLA-matched allogeneic bonemarrow transplantation: a phase I/IIa clinical trial. Biol Blood MarrowTransplant 20:58-65). The assay is based on using flow cytometricevaluation of MHC-class II molecules (HLA-DR) and costimulatory molecule(CD86) expression on iDC membranes after exposure to LPS. As previouslyand repeatedly shown, tolerogenic DCs can be generated upon interactionwith apoptotic cells (Verbovetsky et al., J Exp Med 2002, Krispin etal., Blood 2006), and inhibition of maturation of LPS-treated DCs(inhibition of DR and CD86 expression), occurs in a dose dependentmanner.

During phase 1/2a of the early apoptotic cell clinical study, thepost-release potency assay was conducted for each early apoptotic cellbatch (overall results n=13) in order to evaluate the ability of eachbatch to induce tolerance (Results are shown in FIG. 1 , Mevorach et al.(2014) Biology of Blood and Marrow Transplantation 20(1): 58-65).

DCs were generated for each early apoptotic cell batch from fresh buffycoat, collected from an unknown and unrelated healthy donor, and werecombined with early apoptotic cells at different ratios (1:2, 1:4 and1:8 DC:Early apoptotic Cells, respectively). After incubation with earlyapoptotic cells and exposure to LPS, potency was determined based ondownregulation of DC membrane expression of either HLA DR or CD86 at oneor more ratios of DC: early apoptotic cells. In all 13 assays, earlyapoptotic cells demonstrated a tolerogenic effect, which was seen withpreparations at most DC: early apoptotic cells ratios, and for bothmarkers, in a dose dependent manner.

Monocyte obtained immature DCs (iDCs) were generated from peripheralblood PBMCs of healthy donors and cultured in the presence of 1%autologous plasma, G-CSF and IL-4. iDCs were then pre-incubated for 2hours at 1;2, 1;4 and 1;8 ratios with apoptotic cells either freshlyprepared final product or final product stored at 2-8° C. for 24 hours.The two final products were examined simultaneously in order todetermine whether storage affects potency ability of apoptotic cells.Following incubation, LPS was added to designated wells were left foradditional 24 hours. At the end of incubation, iDCS were collected,washed and stained with both DC-sign and HLA-DR or CD86 in order todetermine changes in expression. Cells were analyzed using flowcytometer and analysis performed using FCS-express software from DC-signpositive cells gate to assure analysis on DCs only.

FIGS. 31A and 31B and FIGS. 32A and 32B show potency test of irradiatedpooled apoptotic cells compared to non-irradiated single donor cell.

Results:

Single Donor Preparations

Table 21 presents the comparative results of non-radiated and irradiatedapoptotic cells; Average cell loss (%) at 24 hours; Annexin positive(⁺)Propidium Iodide (PI) negative(⁻) % at 0 hours and 24 hrs (% of earlyapoptotic cells; Annexin positive (⁺) Propidium Iodide (PI) positive (⁺)% at 0 hours and 24 hrs (% of late apoptotic cells); presence of cellsurface antigens CD3 (T cells), CD19 (B cells), CD56 (NK cells), CD14(monocytes), and CD15^(high) (granulocyte), at 0 hours and 24 hours.

TABLE 21 Final product Apoptotic Cell Apoptotic Cell descriptionApoptotic Cell 2500 rad 4000 rad An⁺PI⁻ t₀ (%) 59.2 59.6 58.4 Range(min-max) (52.6-66.1) (51.6-68.7) (50.4-65.1) An⁺PI⁻ t_(24 h) (%) 62.668.1 66.7 Range (min-max) (53.6-76.3) (52.0-81.3) (52.9-77.1) An⁺PI⁺ t₀(%)  4.9  6.0  6.1 Range (min-max) (3.2-7.0) (5.2-7.4) (4.0-9.1) An⁺PI⁺t_(24 h) (%)  7.3  8.6  9.0 Range (min-max)  (5.0-11.8)  (6.4- 11.8)(6.0-14.9) CD3+ t₀ (%) 56.9 58.3 57.5 Range (min-max) (47.4-66.3)(48.8-67.7) (48.6-66.4) CD3+ t_(24 h) (%) 56.8 57.1 56.6 Range (min-max)(49.6-64.0) (48.0-66.1) (49.7-63.4) CD19+ t₀ (%) 10.6  9.5  9.6 Range(min-max) (10.1-11.0)  (7.7-11.3)  (8.5-10.7) CD19+ t_(24 h) (%) 11.8 9.2  8.8 Range (min-max) (10.2-13.4)  (6.9-11.5)  (7.5-10.1) CD56+ t₀(%) 12.2 13.0 14.4 Range (min-max)  (7.0-17.3)  (7.6-18.4) (21.2-7.6) CD56+ t_(24 h) (%) 12.9 14.1 17.1 Range (min-max)  (8.8-13.4)(10.4-17.8) (10.0-24.1) CD14+ t₀ (%) 23.1 25.2 24.6 Range (min-max)(13.1-33.1) (13.8-36.5) (14.0-35.2) CD14+ t_(24 h) (%) 21.9 23.7 24.4Range (min-max) (13.8-30.0) (13.8-33.6) (15.4-33.4) CD15^(high) t₀ (%) 0.0  0.0  0.01 Range (min-max)  (0.0-0.02) CD15^(high) t_(24 h) (%) 0.0  0.0  0.01 Range (min-max)  (0.0-0.02)

The results in Table 21 show that both non-irradiated apoptotic cellsand irradiated apoptotic cells had comparable percentages of early (rows2 and 3) and late (rows 4 and 5) apoptotic cells. Thus, 25 or 40 Gyirradiation did not accelerate the apoptotic or necrotic process inducedprior to this high level of gamma-irradiation. Further, there wasconsistency between irradiated cell populations vs. controlnon-irradiated population with regard to cell type.

The results of potency assays, presented in FIGS. 31A-31B (HLA-DRexpression) and FIGS. 32A-32B (CD86 expression) show that there was nochange in the immune modulatory capacity of fresh (FIG. 32A, FIG. 32A)and 24 hour-stored (FIG. 31B and FIG. 32B) irradiate apoptotic cellswhen compared with non-irradiated apoptotic cells.

In both FIGS. 31A-31B and FIGS. 32A-32B there is a clear upregulation inboth HLA-DR and CD86 expression, following exposure to maturation agentLPS. Significant (p<0.01), dose-dependent down regulation of bothco-stimulatory markers was observed in the presence of freshly preparedapoptotic cells both from a single donor or irradiated pooled donors. Inaddition, dose dependent down regulation was maintained in both markersin the presence of apoptotic cells stored at 2-8° C. for 24 hours,indicating final product stability and potency following 24 hours ofstorage.

Effect on Dendritic Cells,

In order to test the immunomodulatory capacity of apoptotic cells a postrelease potency assay was used (Mevorach, D., T. Zuckerman, I. Reiner,A. Shimoni, S. Samuel, A. Nagler, J. M. Rowe, and R. Or. 2014. Singleinfusion of donor mononuclear early apoptotic cells as prophylaxis forgraft-versus-host disease in myeloablative HLA-matched allogeneic bonemarrow transplantation: a phase I/IIa clinical trial. Biol Blood MarrowTransplant 20:58-65). No change in immune modulatory assay in dendriticcells was observed. (Data not shown)

Effect on Mixed Lymphocyte Reaction (MLR).

In order to further test the immunomodulatory effect a standardized MLRassay was established. Here, co-cultivation of stimulator and respondercells, i.e. a MLR, yielded strong and reliable proliferation. Uponaddition of non-irradiated apoptotic cells to the MLR, the lymphocyteproliferation was significantly reduced by >5-fold, clearlydemonstrating cell inhibition of proliferation. Inhibition of lymphocyteproliferation in MLRs mediated by irradiated apoptotic cells wascompletely comparable. (Data not shown)

The next step was to evaluate in vivo, irradiated and non-irradiatedapoptotic cells in a completely mismatched mouse model. As shown inFIGS. 28 and 29 , irradiated and non-irradiated early apoptotic cellpreparations had comparable in vivo beneficial effect.

Single Donor Preparations Conclusion:

In conclusion, irradiation of 25 Gy or 40 Gy did not significantlyaccelerate apoptosis or induced necrosis in populations of apoptoticcells. Significantly, these populations maintained the immunomodulatoryeffect of apoptotic cells both in vitro and in vivo.

Multiple Donor Preparations

Next, experiments were performed to verify that the phenomenon observedwith single donor, third party preparation was also true for multiplethird party donors. Unexpectedly, when using pooled individual donorapoptotic cell preparations, the beneficial effect of a single unmatcheddonor was lost (FIG. 30 ). This was not due to GvHD, as the beneficialeffect of each donor separately was maintained (test results no shown).One possibility is that the beneficial effect of the early apoptoticcell preparation was lost due to the interaction of the individual donorcells among themselves. It was further examined whether this possibleinteraction of different donors could be avoided by gamma irradiation.

As shown in FIG. 30 , the beneficial effect of a single donor wascompletely restored following gamma irradiation, wherein the irradiatedmultiple donor preparation and the single donor preparation (irradiatedor non-irradiated) had similar survival patterns.

Conclusion:

It is shown here for the first time that surprisingly irradiation (andpossibly any method leading to T-cell Receptor inhibition) not onlyavoided unwanted proliferation and activation of T-cells but alsoallowed for the beneficial effects of immune modulation when using apreparation of multiple donor third party apoptotic cells.

Example 17: In Vivo Preclinical Analysis of Apoptotic Cell on theTreatment of Sepsis

Objective:

To develop an adjunctive immunomodulating cell-therapy for sepsisprevention of organ failure and mortality in patients with sepsis.Further, to study the effect of early apoptotic cells (Allocetra-OTS)and wide spectrum antibiotics on the course of severe cecal ligationpuncture (CLP) natural history Shown here are the surprising andunexpected effect of early apoptotic cells (Allocetra-OTS), given 4 hafter the end of CLP procedure, in combination with ertapenem antibioticon the development of CLP-induced sepsis in female C57Bl/6 mice. Theeffect was tested in three separated experiments.

Methods:

The Cecal Ligation Puncture (CLP) Mouse Model for Sepsis

The cecal ligation puncture (CLP) model has been proposed to moreclosely replicate the nature and course of clinical sepsis in humans, ascompared to other models, and is considered by many researchers as thegold-standard animal model of sepsis. The CLP model involves theligation of the cecum, usually below the ileocecal junction (to preventbowel obstruction), and at least one centimeter above the distal end ofthe cecum, otherwise the sepsis induced is very mild. The length ofligated cecum, defined as the distance from the distal end of the cecumto the ligation point, determines the severity of the sepsis induced.Following ligation, the cecum is perforated, and this step too can beadjusted to modulate the severity of the sepsis ensuing. The perforationof the cecum is followed by the release of fecal material into theperitoneal cavity to induce a polymicrobial infection, which results inan exacerbated immune response. The benefits of the CLP model are itsreproducibility and potential to alter the severity of sepsis bycontrolling needle size, number of cecal punctures, and antibioticsutilization.

The CLP model was used to evaluate the effect of donor apoptotic cellsthat were shown to have a rebalancing effect on the immune system(Mevorach, D., Zuckerman, T., Reiner, I., Shimoni, A., Samuel, S.,Nagler, A., Rowe, J. M., and Or, R. (2014). Single infusion of donormononuclear early apoptotic cells as prophylaxis for graft-versus-hostdisease in myeloablative HLA-matched allogeneic bone marrowtransplantation: a phase I/IIa clinical trial. Biol. Blood MarrowTransplant. 20, 58-65; Trahtemberg, U., and D. Mevorach. 2017. Apoptoticcells induced signaling for immune homeostasis in macrophages anddendritic cells. Front Immunol 8:1356) in combination with fluidresuscitation and antibiotic treatment.

Study Design

The study reported here summarizes the effect of apoptotic cellsadministered 4 hours after the end of CLP on the development ofCLP-induced sepsis in a female C57BL/6 mouse model.

TABLE 22 Study Design using CLP mouse model Needle Pain FluidsExperiment gauge* Ligation Anesthesia relief reconstitution CLP 19G, 275% Isoflurane. Tramadol 0.5 ml before through above the (100 mg/suturing into and distal end 2 ml). the peritonitis. through *We used75% cecal ligation with 19G needle and two through and throughpuncturing.

TABLE 23 Cohorts Group CLP Allocetra-OTS Ertapenem A + − − B + −(Hartmann) + C + + +

Mice

C57BL/6 female mice, 10-13 wk old, were purchased from ENVIGO (Israel).Mice were kept in an SPF animal facility in compliance withinstitutional IACUC guidelines. Mice were weighed daily and monitored2-3 times a day for clinical signs and determination of the murinesepsis score (MSS) clinical score (according to Shrum, B., Anantha, R.V., Xu, S. X., Donnelly, M., Haeryfar, S. M. M., McCormick, J. K., andMele, T. (2014). A robust scoring system to evaluate sepsis severity inan animal model. BMC Res. Notes 7, 1-11). The endpoint was defined astotal score of 15 or maximum score in one of the categories in thetable.

Cecal Ligation and Puncture (CLP) Procedure

The procedure was performed as briefly described below: Cecal Ligationand Puncture (CLP)—Experimental method. Briefly, the mice were operatedunder isoflurane anesthesia machine. Mice were anesthetized by 4%isoflurane in the chamber for about 1 minute, following anesthesia micewere transferred to the operation table and connected to the isofluranenuzzle with 2% isoflurane. Mice were administered analgesics bysub-cutaneous (S.C.) injection of tramadol 5 mg/kg in 0.1 ml ofpre-warmed 0.9% saline solution. After opening the abdomen of the mouseby a midline incision, the cecum was exposed. The cecum was ligated 75%above its distal end with a 4-0 silk suture. Following ligation, thececum was perforated twice with a 19-gauge needle, using thethrough-and-through technique (introducing a needle through the cecum).The perforation of the cecum was followed by the release of fecalmaterial into the peritoneal cavity. Afterwards, the cecum was returnedto the peritoneal cavity and 0.5 ml of pre-warmed saline wasadministered to the peritoneal cavity, which was subsequently suturedwith a 4-0 vicryl suture. The skin was then closed with 9 mm clips andthe mice were placed under a heating lamp to recuperate. Mice receivedtramadol every 12 h for the first 36 h after the procedure. In certainruns of the in vivo experiment, mice received the second dose oftramadol in 100 μl of saline and in other runs of the in vivoexperiments, the mice received the second dose of tramadol in 0.5 mlsaline in order to add fluid as supportive care. Mice that died duringthe first 24 hours after surgery were considered as perioperativemortality and were immediately excluded from the experiment, as theirdeath was due to perioperative complications and not to sepsis.

Allocetra-OTS (Irradiated Early Apoptotic Cells)

Enriched mononuclear cell fraction was collected via Leukapheresisprocedure from healthy, eligible donors. Following apheresis completion,cells were washed and resuspended with freezing media composed ofPlasmaLyte A pH 7.4, 5% Human Serum Albumin, 10% dimethyl sulfoxide(DMSO), 5% Anticoagulant Citrate Dextrose Solution-Formula A (ACD-A) and0.5 U\ml heparin. Cells were then gradually frozen and transferred toliquid nitrogen for long term storage.

For preparation of Allocetra-OTS, cryopreserved cells were thawed,washed and resuspended with apoptosis induction media, composed of RPMI1640 supplemented with 2 mM L-glutamine and 10 mM Hepes, 10% autologousplasma, 5% ACD-A, 0.5 U\ml heparin sodium and 50 μg/mlmethylprednisolone. Cells were then incubated for 6 hours at 37° C. in5% CO₂. At the end of incubation, cells were collected, washed andresuspended in Hartmann's solution using the LOVO cell processing system(Fresenius Kabi, Germany). Following manufacturing completion, Allocetrawas irradiated at 4000 cGy at the radiotherapy unit (Gammacell 220excel, MDS nordion), Hadassah Ein Kerem Medical center, Jerusalem,Israel. Allocetra-OTS cells were centrifuged at 290 g, for 10 min at2-8° C., and resuspended in Hartmann's solution for injection.

Apoptosis and viability of Allocetra-OTS were determined using AnnexinVand PI staining (MBL, MA, USA) by flow-cytometry (FACSCalibur, BD).Results analyzed using FCS express software. Cells were >50% An⁺PI⁻ and<7% An⁺PI⁺.

In some cases, variable amounts of Allocetra-OTS cells were injectedi.v. per mouse 4 h after the end of CLP procedure. In other instances,20×10⁶ Allocetra-OTS cells were injected IV per mouse. The CLPprocedures required about 20 minutes per mouse and the overall procedurelasted for about 5 hours. Allocetra-OTS was injected into each mouse 4 hafter its procedure had ended. Control mice received Hartmann vehiclesolution at the same time point. For dose calibration of Allocetra-OTScells, each mouse received 1, 3, 6, 10, or 20×10⁶ cells. Control micereceived Hartmann vehicle solution.

Antibiotic Treatment

Mice received 75 mg/kg Ertapenem i.p immediately after Allocetra-OTSadministration and then every 24 h for 3 days.

Serum Cytokines/Chemokines.

At the indicated times (pre-CLP, 24 h, 48 h, and 72 h post-CLP) ˜500 μlblood was collected in a pre-labeled Eppendorf tube and left for 30 minto allow clotting. The samples were centrifuged at 1800 g (3000 rpm) for10 min at 4° C., 200 al. Serum was transferred to a new pre-labeledEppendorf tube and kept at 4° C. Excess serum was stored at −80° C.Cytokine/chemokine measurement was performed using the Luminex MAGPIXsystem, and analysis was performed with Milliplex software.

Organ Dysfunction Tests.

22-24 h after CLP, mice were weighed, assigned an MSS clinical score andsacrificed for the evaluation of organ dysfunction. Mice were bledthrough the retro-orbital sinus (venous blood). Naïve mice were bledunder isoflurane analgesia; CLP mice were bled without analgesia due tothe concern of death.

Blood Pressure.

Mice were measured for blood pressure using a CODA noninvasive bloodpressure system (Kent scientific corporation). Blood pressure wasmeasured the day before the CLP procedure, in order to establish abaseline, and every 4 hours following the CLP procedure. For each mouse,3 measurements were made each time blood pressure was assessed toprovide more accurate data.

Blood gas.

100 μl blood was collected using heparin-coated capillary tube (PaulMarienfeld, K G, Lauda-Knigshofen, Germany) and immediately tested usingSTAT profile prime machine (Nova Biomedical, Waltham, Mass., USA).

Hematology.

2501 blood was collected into EDTA tubes (MiniCollect, Greiner Bio-One,Kresmtinster, Austria), tubes were rotated to prevent blood clotting andkept at 4° C. Hematology analysis was performed by AML laboratories(Herzliya, Israel).

Biochemistry.

About 500 μl blood was collected to a pre-labeled Eppendorf tube andleft for −30 min to allow clotting. The samples were centrifuged at 1800g (3000 rpm) for 10 min at 4° C., 200 μl serum was transferred to a newpre-labeled Eppendorf tube and kept at 4° C. Excess serum was stored in−80° C. Biochemistry analysis was performed by AML laboratories(Herzliya, Israel).

Lungs.

24 hours post-CLP, mice were weighed and then sacrificed; lungs wereharvested and weighed, and lung-to-body weight ratios were calculated.

NGAL, Cystatin C, Complement (C5a, C3a).

Blood was collected to pre-labeled Eppendorf tubes and left for −30 minto allow clotting. Tubes were centrifuged at 1800 g (3000 rpm) for 10min at 4° C. Serum was transferred to new pre-labeled Eppendorf tubesand stored at −80° C. for Luminex (NGAL and Cystatin C) and ELISA (C3aand C5a) evaluation.

Luminex® Analysis.

Cytokine/chemokine measurement was performed using the Luminex MAGPIXsystem, and analysis was performed with Milliplex software. NGAL andCystatin C were tested by Luminex Multiplex kit (Millipore,MKI2MAG-P4k). All reagents were provided with the kit, and all reagentswere prepared according to the manufacturers' protocols. The assays wereperformed in 96-well plates according to the protocol provided. Platereading was performed with the Luminex MAGPIX system (Luminex Corp.) andanalyzed using Milliplex software (Millipore). The analysis software wasset to acquire data using 50 μl of sample per well, to collect not lessthan 50 beads (range 200-800 events per single bead set). The raw datawas measured as mean fluorescence intensity (MFI) and the concentrationof each analyte for each sample was calculated using a 4- or 5-parameterlogistic fit curve generated for each analyte from the 7 standards. Thelower limit of quantification (LLOQ) was determined using the loweststandard that was at least 3 times above background. The calculation ofthe LLOQ was performed by subtracting the MFI of the background(diluent) from the MFI of the lowest standard concentration andback-calculating the concentration from the standard curve.

ELISA.

Complement components were tested by sandwich ELISA kits: C3a (TECO,TEI038) and C5a (EA100633, OriGene, Rockville, Md., USA). All reagentswere provided with the kits and prepared according to the manufacturers'protocols. Assays were performed in 96-well plate according to theprotocols provided. OD plate reading was performed with the Infinite F50(TECAN, Mainnedorf, Switzerland) and analyzed using Magellan software(TECAN). The raw data was measured as 450 nm optical density (OD) andthe concentration was calculated using a linear standard curve generatedfrom 6-7 standards. The lower limit of quantification (LLOQ) wasdetermined using the lowest standard. The calculation of the LLOQ wasperformed by subtracting the OD of the background (diluent) from the ODof the lowest standard concentration and back-calculating theconcentration from the standard curve.

2D Echocardiography.

24 hours after CLP, naïve mice (n=5) or Ertapenem-treated CLP mice(n=10) were anesthetized with isoflurane and their left ventricle (LV)was imaged by echocardiography using a high-resolution imaging system(Vevo 770, Visual Sonics, Canada). LV internal distances, heart rate,and posterior wall thickness were measured for the calculation ofvarious parameters of LV structure and function. LV volume and ejectionfraction (EF) were calculated using the Teichholz method, and relatedparameters were calculated as previously described (Stypmann et al.,2009).

Bioenergetics Analysis. Cell Isolation, Seeding, and Analysis.

24 hours after CLP, mice were euthanized, the spleen was extracted andsplenocytes were dissociated. cells were seeded at a density of 0.5×10⁶cells/well into XF96 well plates pre-coated with poly-D-lysine (100μg/mL) to maximize adherence and allowed to adhere overnight. Afterrecording of basal measurements, the Mito Stress Test (Agilent, SantaClara, Calif., USA) injection strategy consisted of oligomycin (1 PM),FCCP (1 NM), and rotenone/antimycin A in combination (1 μM). TheGlycolytic Stress Test (Agilent) injection strategy consisted of glucose(10 mM) and oligomycin (1 PM), followed by 50 mM 2-deoxyglucose (2DG).Oxygen consumption rate (OCR) and extracellular acidification rate(ECAR) were measured with the XF96 Extracellular Flux Analyzer (SeahorseBioscience, North Billerica, Mass., USA) using three 3 min cycles of mixand measurement following each injection. Normalization: Upon completionof the extracellular flux assay, plated cells were lysed, and theirprotein concentrations were quantified using the BCA assay. Briefly,cells were lysed with 50 μl RIPA lysis medium supplemented with proteaseinhibitors for each well and agitated for 5 min, cells were incubated atRT for 30 min, and after incubation lysate samples were added to BCAworking reagent medium and measured for absorbance at 562 μm.

Data Analysis:

Assay data were analyzed with MS Excel, using the XF Report Generator,macro-enabled spreadsheet (Agilent).

Statistical Method

Unless differently indicated, data are presented as the median and theerror bars represent the 5-95 percentile range. Differences betweengroups were examined for statistical significance using the Mann-Whitneynonparametric test. Differences between multiple groups were examinedfor statistical significance using Kruskal-Wallis one-way analysis ofvariance (non-parametric ANOVA) with multiple-comparisons adjusted byusing the Dunn's test. Lung/body weight ratio was examined using one-wayanalysis of variance (ANOVA). Correlation of any parameter to clinicalscore was evaluated by a Spearman's rank correlation coefficient, with acoefficient higher than 0.7 or lower than −0.7 being a strongcorrelation. All statistical analyses were done using GraphPad Prism.Survival analysis was performed according to the Kaplan-Meier method.Log rank statistical test was performed using GraphPad (CA, USA).

Results:

The effect of Allocetra-OTS, given 4 hours after the end of CLPprocedure, in combination with the ertapenem antibiotic, a highlyeffective antibiotic commonly used for the treatment of severe orhigh-risk bacterial infections, including urinary or abdominalinfections, was evaluated in several studies. Mice were weighed dailyand monitored two to three times per day for clinical signs anddetermination of the murine sepsis score. The endpoint was defined assurvival (either death or sacrifice when a total clinical score of 15 ormaximum score in one of the categories was reached).

Evaluation of the MSS Clinical Scoring System as a Surrogate Indicatorfor Organ Dysfunction in CLP Mice.

Sepsis elicits dysregulated immune responses, which in turn dramaticallydisrupt the physiological homeostasis of vital organs including thekidney, liver, lungs, and heart. This imbalance often rapidly escalatesinto Multiple organ dysfunction syndrome (MODS), which is usuallyassociated with poor outcomes. A MODS-like disease has been previouslyreported in murine CLP models (Coletta, C., Mòdis, K., Oláh, G.,Brunyánszki, A., Herzig, D. S., Sherwood, E. R., Ungviri, Z., and Szabo,C. (2014). Endothelial dysfunction is a potential contributor tomultiple organ failure and mortality in aged mice subjected to septicshock: preclinical studies in a murine model of cecal ligation andpuncture. Crit. Care 18, 511; Drechsler, S., Weixelbaumer, K. M.,Weidinger, A., Raeven, P., Khadem, A., Redl, H., van Griensven, M.,Bahrami, S., Remick, D., Kozlov, A., et al. (2015). Why do they die?Comparison of selected aspects of organ injury and dysfunction in micesurviving and dying in acute abdominal sepsis. Intensive Care Med. Exp.3, 48; Osterbur, K., Mann, F. A., Kuroki, K., and DeClue, A. (2014).Multiple organ dysfunction syndrome in humans and animals. J. Vet.Intern. Med. 28, 1141-1151; Ruiz, S., Vardon-Bounes, F., Merlet-Dupuy,V., Conil, J.-M., Buleon, M., Fourcade, O., Tack, I., and Minville, V.(2016). Sepsis modeling in mice: ligation length is a major severityfactor in cecal ligation and puncture. Intensive Care Med. Exp. 4, 22;Seemann, S., Zohles, F., and Lupp, A. (2017). Comprehensive comparisonof three different animal models for systemic inflammation. J. Biomed.Sci. 24); however, histopathological analysis of organ dysfunction maynot be an effective research tool for the development of therapeuticapproaches in this model because it is a terminal procedure, requiring alarge number of mice. In addition, histopathological results often showno differences between experimental groups and fail to correlate withdisease severity and outcomes. Thus, finding diagnostic tests for organdysfunction in septic

mice that strongly correlate with the MSS clinical score may be aclinically relevant research tool for sepsis.

Therefore, 24 hours post-CLP, each mouse (Fluids and Ertapenem-treated;N=40) was assigned with a clinical score, weighed, and blood sampleswere drawn from the retro-orbital sinus for further analyses. Mice weresacrificed and their lungs were harvested and weighed. To elucidate theeffects of CLP on organ dysfunction and its correlation with the MSSclinical score, blood was tested for multiple parameters of organdysfunction relating to five major systems: cardiovascular, respiratory,renal, hepatic, and hematological, as well as complement and severalmetabolites (Tables 24A and 24B).

TABLE 24A Organ dysfunction analysis 24 hours post CLP. ³AUC²Correlation of Median of Median of CLP + ¹P- to Clinical ROC SystemParameter Naïve [IQR] Ertapenem[IQR] Value Score Curve Respiratoryvenous 44.75 [42.0, 59.28]; N = 8 53.2 [43.4, 79.8]; N = 11 n.s No N/ApCO2 (mmHg) venous pO2 46.45 [39.15, 59.98]; N = 8 57.4 [51.1, 63.3]; N= 13 n.s 0.5934 N/A (mmHg) ‡pH 7.3245 [7.31, 7.341]; N = 9 7.1995 [7.02,7.274]; N = 12  0.0089 −0.7792 0.8438 ‡Lung/body 0.0069 [0.0065, 0.007];N = 21 0.0079 [0.0071, 0.0089]; N = 40 <0.0001 0.743 0.8494 weight (w/w)Renal Creatinine 0.17 [0.145, 0.205]; N = 9 0.16 [0.11, 0.31]; N = 15n.s No N/A (mg/dL) ‡urea 37.1 [33.9, 42.5]; N = 9 116.6 [67.8, 789.1]; N= 15 <0.0001 0.8852 1 (mg/dL) Cystatin C 650 [600, 700]; N = 4 750 [525,1575]; N = 16 n.s 0.6196 N/A (ng/ml) ‡NGAL 150 [100, 275]; N = 4 35850[23350, 50975]; N = 16  0.0004 0.7572 1 (ng/ml) Hepatic ‡total 5.54[5.405, 5.61]; N = 9 4.14 [3.77, 4.32]; N = 15 <0.0001 −0.865 1 protein(g/dL) ‡Albumin 4.2 [4.05, 4.3]; N = 9 2.9 [2.6, 3.1]; N = 15 <0.0001−0.8333 1 (g/dL) Globulin 1.33 [1.235, 1.41]; N = 9 1.26 [1.12, 1.36]; N= 15 n.s −0.5312 N/A (g/dL) ‡AST (U/L) 345 [290, 519]; N = 9 1003 [873,1328]; N = 15 <0.0001 0.7268 0.9852 ‡ALT 133 [94.5, 197.5]; N = 9 374[327, 502]; N = 15 <0.0001 0.8216 0.9926 (U/L) ‡Alkaline 192 [169, 202];N = 9 101 [93, 110]; N = 15 <0.0001 −0.8432 1 Phosphatase (U/L) total0.09 [0.065, 0.1]; N = 9 0.09 [0.07, 0.12]; N = 15 n.s No N/A Bilirubin(mg/dL) ‡Significant differences between CLP mice and naïve mice with astrong correlation to MSS Clinical score (−0.7 > ρ- Spearman > 0.7)¹Mann-Whitney 2-tailed nonparametric t-test; ²ρ- Spearman; ³Naïve versusCLP mice

TABLE 24B Organ dysfunction analysis 24 hours post CLP. ³AUC²Correlation of Median of Median of CLP + ¹P- to Clinical ROC SystemParameter Naïve [IQR] Ertapenem [IQR] Value Score Curve Hematopoietic‡WBC 2.585 [2.19, 3.605]; N = 8 1.94 [1.06, 2.18]; N = 15 0.0017 No0.8833 (10³/μL) RBC 9.935 [8.28, 10.17]; N = 8 8.36 [8.12, 9]; N = 15n.s No N/A (10⁶/μL) Hemoglobin 14.85 [12.48, 15]; N = 8 12.4 [12, 14]; N= 15 n.s No N/A (g/dL) HCT (%) 46.4 [41.55, 47.7]; N = 8 40.9 [39.3,43.1]; N = 15 n.s No N/A MCV (fL) 47.7 [46.25, 50.45]; N = 8 47.9 [46.3,49.4]; N = 15 n.s No N/A MCH (pg) 14.95 [14.75, 15.1]; N = 8 14.9 [14.7,15.3]; N = 15 n.s No N/A MCHC 31.7 [30.23, 32.28]; N = 8 31.2 [30.5,33.3]; N = 15 n.s No N/A (g/dL) ‡Platelets 642.5 [548.5, 812.8]; N = 899 [87.25, 228.5]; N = 14 <0.0001  −0.7099 1    (10³/μL) Neutrophils0.51 [0.3175, 0.8125]; N = 8 0.31 [0.17, 0.39]; N = 15 0.0382 −0.4531N/A (10³/μl) Lymphocytes 1.965 [1.723, 3.175]; N = 8 1.55 [0.68, 1.9]; N= 15 0.0275 No N/A (10³/μl) Monocytes 0 [0, 0]; N = 8 0 [0, 0.04]; N =15 N/A No N/A (10³/μl) Eosinophils 0 [0, 0]; N = 8 0 [0, 0]; N = 15 N/AN/A N/A (10³/μl) Basophils 0 [0, 0]; N = 8 0 [0, 0]; N = 15 N/A N/A N/A(10³/μl) Complement ‡C3a (ng/ml) 8903 [7769, 11426]; N = 4 4835 [4216,5652]; N = 16 0.0029 −0.7183 0.9531 C5a (ng/ml) 1102 [992, 1664]; N = 41809 [1631, 2492]; N = 16 0.0219 No 0.875  Metabolites Cholesterol 97[92.5, 100]; N = 9 108 [88, 140]; N = 15 n.s No N/A (mg/dL) Glucose 151[118.5, 178]; N = 17 59.5 [42.75, 122.3]; N = 28 0.0001 −0.3227 0.8288(mg/dL) Lactate 19.5 [17.25, 24.75]; N = 8 13 [9.5, 20]; N = 13 0.0255No N/A (mg/dL) Electrolytes Phosphorus 8.8 [8.3, 9.1]; N = 9 10.2 [8.5,13.7]; N = 15 n.s  0.6705 N/A (mg/dL) Sodium 151 [147.8, 153]; N = 17154.1 [150.1, 158]; N = 26 0.0125 No N/A (mmol/L) Potassium 5.7 [5.225,5.9]; N = 17 6.6 [6.1, 7.3]; N = 27 0.0001  0.4993 0.8279 (mmol/L)Chloride 115.6 [113.5, 117]; N = 17 121.1 [119, 125.3]; N = 27 <0.0001  0.5063 0.939  (mmol/L) ‡Significant differences between CLP mice andnaïve mice with a strong correlation to MSS Clinical score (−0.7 > ρ-Spearman > 0.7) ¹Mann-Whitney 2-tailed nonparametric t-test; ²ρ-Spearman; ³Naïve versus CLP mice

CLP mice were compared to naïve mice (MSS score of 0; N=21). The CLPmice were divided into three sub-groups, based on their clinical scores:1-4 (mild sepsis), 7-12 (moderate sepsis), and 13+ (severe sepsis). 24hours post-CLP, most mice exhibited severe clinical signs, with a medianMSS clinical score of 13 (95% CI of 9-14), indicating moderate to severesepsis (FIG. 34A).

To study cardiac function of CLP-mice, 24 hours post-CLP, the leftventricle (LV) of naïve mice (n=5) or Ertapenem-treated CLP mice (n=10)was imaged by echocardiography and various structural and functionalcardiac parameters were tested for their correlation with the clinicalscore (Table 25).

TABLE 25 2D Echocardiography parameter analysis ²Correlation Median ofNaïve Median of CLP ¹P- to Clinical Parameter [IQR] [IQR] Value Score^(‡)Heart rate (BPM); 500 [458, N = 5 358.5 [270, N = 10 0.003 −0.878 HR561]; 409]; ³Fractional 30 [28.45, N = 5 40.4 [30.6, N = 9  n.s Noshortening (%); FS 40.8]; 51.6]; ⁴Ejection fraction 57.7 [55.3, N = 571.9 [59.1, N = 9  n.s No (%); EF 71.45]; 84.15]; Posterior wall 25[19.55, N = 5 15 [5.3, N = 9  n.s No thickness (mm); 37.65]; 50.25]; PWT⁵LV Volume- 68.3 [56.8, N = 5 39.1 [26.9, N = 9  0.0035 −0.701 diastole(μl); 82.7]; 50.05]; LVEDV ⁵LV Volume-systole 29 [17.35, N = 5 11.8[4.2, N = 9  0.018 −0.597 (μl); LVESV 37.05]; 17.25]; LV Area-diastole10.3 [9.4, N = 5 8.44 [5.95, N = 9  0.002 No (mm²); LVEDA 11.58]; 8.72]LV Area-systole 5.15 [4.62, N = 5 3.57 [2.09, N = 9  0.042 No (mm²);LVESA 5.94]; 4.76]; ⁶Fractional area 54.42 [26.32, N = 5 54.54 [40.37, N= 9  n.s No shortening (%); FAS 58.36]; 70.93]; ⁷LV Stroke volume 41.3[38.5, N = 5 23.4 [21.25, N = 9  0.007 −0.691 (μl); SV 45.65]; 37.5];^(8,‡)Cardiac output 20.62 [18.41, N = 5 9.34 [7.33, N = 9  0.002 −0.799(ml/min); CO 24.6]; 11.92]; LV internal distances (diastole/systole,LVIDD, and LVIDS, respectively), HR and PWT were measured in duplicatesor triplicates using the M-Mode view of echocardiograms; LVEDA and LVESAwere measured using the B-mode view of the echocardiograms.^(‡)Significant differences between CLP mice and naïve mice with astrong correlation to MSS Clinical Score (−0.7 > ρ-Spearman > 0.7)¹Mann-Whitney 2-tailed nonparametric t-test; ²ρ-Spearman; ³${{FS}(\%)} = {\frac{{LVIDD} - {LVIDS}}{LVIDd} \times 100\text{;}}$ ⁴${{EF}(\%)} = {\frac{{LVEDV} - {LVESV}}{LVEDV} \times 100}$ ⁵Teichholzmethod for LV volume calculation:${{LVEDV}\left( {\mu\; l} \right)} = {\frac{7 \times {LVIDD}^{3}}{\left\lbrack {2.4 + {LVIDD}} \right\rbrack}\text{;}}$${{LVESV}\left( {\mu\; l} \right)} = {\frac{7 \times {LVIDS}^{3}}{\left\lbrack {2.4 + {LVIDS}} \right\rbrack}\text{;}}$⁶ ${{FAS}(\%)} = {\frac{{LVEDA} - {LVESA}}{LVEDA} \times 100\text{;}}$⁷SV(μl) = LVEDV − LVESV; ^(8,‡)${{CO}\left( \frac{ml}{\min} \right)} = {{SV} \times {HR}\text{/}1000}$

Severe Cardiovascular and Respiratory Dysfunction in CLP Mice.

The cardiovascular system is among the first to be affected in mice withCLP-induced sepsis. Accordingly, the attempts at non-invasivemeasurement of the murine blood pressure were not successful because thesystolic blood pressure was below the instrument's detection limit of<50 mmHg, further emphasizing the severity of sepsis. Lung dysfunctionwas evident by the increased lung weight (normalized to body weight),due to fluid retention; this significant increase of lung weightstrongly correlated with the MSS clinical score (Table 24; ρSpearman=0.743), and accordingly was even more significant in mice withmoderate and severe sepsis (FIG. 34B; p≤0.01 and p≤0.0001 for MSSclinical scores of 7-12 and 13+, respectively). Though CLP mice had noapparent structural myocardial damage (FIG. 34C, top view; B-Mode) theyhad a significantly lower heart rate (FIG. 34C, M-Mode) than naïve mice,with a strong inverse correlation to the MSS clinical score (Table 25; pSpearman=−0.878); this reduced heart-rate was most significant in micewith severe sepsis (FIG. 34D; p≤0.0194). Although the fractionalshortening (FS) and ejection fraction (EF) were not significantlydifferent between the groups (Table 25; p=n.s), CLP-mice hadsignificantly lower diastolic LV volume, with a strong inversecorrelation to clinical score (Table 25; ρ Spearman=−0.701); severelyseptic mice had the lowest LV volume (FIG. 34E; p≤0.0343). The systolicLV volume and the measured LV area were also significantly lower inseptic mice (Table 25). Accordingly, cardiac output of CLP mice wasseverely impaired and, again, strongly and inversely correlated withdisease severity (Table 25, ρ Spearman=−0.799 and FIG. 34F).

Acute Kidney Injury (AKI).

An exaggerated inflammatory response combined with cardiovasculardysfunction in sepsis can seriously damage renal function. Therefore,renal dysfunction was evaluated by measuring creatinine and urea, aswell as newer markers, i.e. cystatin C and NGAL. Though slightlyelevated, CLP mice had no significant increase in serum creatinine andcystatin C (Tables 24A and 24B), indicating probably a relatively lateeffect on creatinine levels. However, urea levels were significantlyelevated in CLP mice with low (1-4) and moderate (7-12) clinical score(FIG. 35A; p<0.01 for both groups), and strongly correlated with MSSclinical score (Tables 24A and 24B; ρ Spearman=0.8852). In contrast tothe late effect on serum creatinine, NGAL was suggested to correlatewell to AKI in sepsis model mice (Otto, G. P., Busch, M., Sossdorf, M.,and Claus, R. A. (2013). Impact of sepsis-associated cytokine storm onplasma NGAL during acute kidney injury in a model of polymicrobialsepsis. Crit. Care 17, 419). Indeed, NGAL serum concentration wasdramatically increased, especially in CLP mice with severe sepsis (FIG.35B; p≤0.01; MSS clinical score of 13+), and strongly correlated withthe clinical score (Tables 24A and 24B; ρ Spearman=0.7572). Togetherwith a moderate but significant increase in serum potassium in CLP mice(FIG. 35C; p≤0.01; MSS clinical score of 13+), these results areindicative of AKI.

Markers for acute liver injury strongly correlate with MSS clinicalscore in CLP mice. Liver dysfunction occurs in almost 40% of sepsispatients; it can be diagnosed by an increase of serum bilirubin andliver transaminases, and a decrease in protein production, includingalbumin. CLP mice were shown to follow the same trend. In this study,CLP mice with severe sepsis had a mild but insignificant increase ofserum bilirubin (FIG. 36A, p>0.93). Nevertheless, both AST and ALTtransaminase levels were significantly elevated in CLP mice, compared tonaïve mice (Tables 24A and 24B; p≤0.001), especially in mice with severesepsis (FIGS. 36B and 36C; p≤0.01). The dramatic increase in AST and ALTwere clearly reflected in murine MSS clinical scores (Tables 24A and24B; ρ Spearman=0.7268 and 0.8216, respectively). A substantial releaseof liver transaminases that is not accompanied by significant increaseof bilirubin is typical of hypoxic hepatitis and may suggest thismechanism of ALI. Alkaline phosphatase (ALP) is also elevated humansepsis patients, possibly as an anti-inflammatory and anti-microbialagent with a protective function against acute kidney injury. Indeed, insevere sepsis as in this model and with severe AKI, ALP serumconcentration in CLP mice was substantially reduced in comparison tonaïve mice, and with a strong inverse correlation to MSS clinical score(Tables 24A and 24B; p<0.0001, p Spearman=−0.8432). This reduction ofALP was most prominent in mice with moderate and severe sepsis (FIG.36D; p≤0.017 and p≤0.001 for MSS clinical scores of 7-12 and 13⁺,respectively).

AP are endogenous metalloenzymes found in serum and in multiple organsthroughout the body including bone, liver, intestine, and kidney. Theseenzymes are well established as biomarkers of liver and bone disease,but their physiologic roles remain incompletely understood. Recentevidence points towards a potential protective effect of AP in themitigation of AKI through dephosphorylation of nephrotoxic moleculesincluding extracellular adenine nucleotides and endotoxin. Less is knownabout ALP serum concentration in CLP mice, although a few studiesdemonstrated an increase of ALP following CLP in mice, the oppositeobservation seen here seems to reflect the severity of CLP. 24 hourspost-CLP, both total protein serum levels, and serum albumin levels hadsignificantly dropped (Tables 24A and 24B; p<0.0001); these decreasedprotein levels are probably attributed to liver dysfunction, as albumin(which is produced primarily in the liver), but not globulin, wasdecreased (FIGS. 36E and 36F). Interestingly, glucose levels weresignificantly decreased, mainly in mildly septic mice (FIG. 36G; p≤0.01for MSS clinical scores of 1-4), but also in general (Tables 24A and24B; p≤0.0001). This phenomenon may be related to liver dysfunction ofgluconeogenesis.

Marked Thrombocytopenia and Lymphopenia in Septic Mice.

The hematological system is the first and one of the most affected insepsis. Hematological aberrations in sepsis patients includethrombocytopenia, lymphopenia) and neutropenia or neutrophilia; all ofwhich are associated with poor outcomes. CLP mice are no different.Hematological dysfunction in septic mice was thus evaluated by fullblood count, including red blood cells (RBC), platelets, white bloodcells (WBC, both general and sub-populations), and other parameters(hemoglobin, hematocrit, and cell volume). As seen in Tables 24A and24B, the most dramatic effect on the hematological system was a sharpdecrease (−6.49×fold change) of CLP-mice platelet count, in comparisonto naïve mice (p<0.0001). This thrombocytopenia was in strongcorrelation to MSS clinical score (ρ Spearman=−0.7099), and moreprominent in mice with moderate and severe sepsis, with median plateletcounts below 100×10³/μl (FIG. 37A; 95% CI range of 37-260 10³/μl). Therewere no differences between CLP-mice and healthy mice in RBC,hemoglobin, hematocrit, and cell volume, (Tables 24A and 24B; p=NS). Aslight neutrophilia was also observed in septic mice, with a moderateinverse correlation to clinical score (Tables 24A and 24B; p≤0.0382; ρSpearman=−0.4531). However, total WBC count in septic mice wassignificantly lower than that in healthy mice (Tables 24A and 24B;p≤0.0017) and with possibly inverse correlation to MSS clinical score.Interestingly, the lowest WBC count was in mice with mild sepsis ratherthan mice with severe sepsis (FIG. 37B; p≤0.01 for MSS clinical scoresof 1-4). This was mainly due to lymphocytes that were with lower countsin septic mice (Tables 24A and 24B; p≤0.0275), which was mainlyattributed to severe lymphopenia in mildly septic mice (FIG. 37C; p≤0.01for MSS clinical scores of 1-4).

Aberrant Complement Activation Pattern Following CLP.

The complement immune system is a major responder to infection, and assuch is highly activated in sepsis. The effect of sepsis on thecomplementary immune system was evaluated by measuring the serumconcentration of C3a and C5a, 24 hours post-CLP. As expected, C5a serumconcentrations were elevated in CLP-mice; however, they did notcorrelate with MSS clinical score (Tables 24A and 24B; p≤0.0219). Asseen in FIG. 37D, C5 was active in all CLP-mice, regardless of theirclinical score. Interestingly, C3a levels were significantly decreasedin CLP-mice, and strongly correlated with MSS clinical score (Tables 24Aand 24B; p≤0.0029, ρ Spearman=−0.7183); This decrease was the mostsignificant in mice with a severe clinical score (FIG. 37E; p≤0.012 forMSS clinical scores of 13+).

CLP Mice Presented Adverse Metabolic Changes.

Because the pathogenesis of sepsis involves dramatic metabolic changes,it is of interest to explore some of the major metabolic andbioenergetic markers of sepsis and to find their correlation to diseaseseverity. CLP mice had a significantly lower blood pH than naïve mice,with a strong inverse correlation to clinical score (Tables 24A and 24B;p≤0.0089, ρ Spearman=−0.7792); the median blood pH of mice with severesepsis, compared to naïve mice, was even lower (FIG. 38A; 7.044 and7.325, respectively), suggesting respiratory or metabolic acidosis. Asnoted above (FIG. 36G), glucose levels were significantly decreased inseptic mice, which may also be related to a state of hypoxia (whereasglucose is rapidly catabolized in the glycolysis pathway). In order tofurther explore these phenomena, bioenergetics analysis was performedusing the XF96 Extracellular Flux Analyzer (Seahorse Bioscience, NorthBillerica, Mass., USA) to measure the oxygen consumption rate (OCR) andextracellular acidification rate (ECAR) of freshly isolated PBMCs fromnaïve and CLP-mice. These levels directly reflect mitochondrial functionand glycolysis. The general mitochondrial respiration of PBMCs fromCLP-mice was compromised, especially in mice with severe clinical scores(FIG. 38B), as manifested by significantly decreased maximal respiration(P<0.05, Table 3), a mildly increased proton leak, and reduced sparerespiratory capacity (p=n.s, Table 3).

TABLE 26 Mitochondrial function and glycolysis assay analysis of micederived splenocytes, 24 h post-CLP (N = number mice) ²Correlation Medianof Median of ¹P- to Clinical *Assay Parameter Naïve [IQR] CLP [IQR]Value Score Mitochondrial Non- 5.77 [5.16, 5.86]; N = 3 4.57 [4.09,5.26]; N = 7 0.0667 −0.8247 Respiration mitochondrial (OCR) oxygenconsumption Basal 14.78 [12.72, 16.08]; N = 3 13.88 [11.89, 14.59]; N =7 0.3833 No respiration Maximal 39.94 [25.74, 45.41]; N = 3 26.55 [24.5,33.16]; N = 7 0.1833 No respiration Proton leak 3.33 [2.06, 3.47]; N = 34.1 [3.25, 4.22]; N = 7 0.1833 No ATP 11.31 [10.67, 12.75]; N = 3 9.68[8.9, 10.15]; N = 7 0.0667 −0.7547 production Spare 23.87 [13.01,30.63]; N = 3 12.61 [10.7, 19.23]; N = 7 0.1167 −0.8528 respiratorycapacity Spare 249 [200, 308]; N = 3 204 [184, 234]; N = 7 0.2667−0.8528 respiratory capacity as a % Coupling 79.2 [76.3, 83.6]; N = 369.7 [69, 72.9]; N = 7 0.0167 −0.6688 efficiency (%) Glycolytic Non-0.89 [0.82, 1]; N = 3 0.92 [0.79, 0.98]; N = 7 >0.9999 No functionglycolytic (ECAR) acidification Glycolytic 0.54 [0.41, 0.81]; N = 3 0.81[0.6, 0.83]; N = 7 0.25 No capacity Glycolysis 1.24 [1.09, 1.25]; N = 31.05 [0.87, 1.31]; N = 7 0.5167 No Glycolytic 0.68 [0.43, 0.71]; N = 30.35 [0.18, 0.50]; N = 7 0.2667 −0.7301 reserve Glycolytic 232 [157,268]; N = 3 142 [124, 175]; N = 7 0.1167 −0.8528 reserve as a %

Significantly reduced ATP production and coupling efficiency were alsostrongly and inversely correlated with the MSS clinical score (Table 26;p≤0.001, ρ Spearman <−0.7 and FIG. 38C). ECAR analysis of the same cellsrevealed very mild changes in glycolytic function, changes that wereseen only in moderately septic mice, which had slightly increasedglycolysis (FIG. 38D, MSS clinical scores of 7-8.5). The only glycolyticparameter that was in correlation with the clinical score was theglycolytic reserve (Table 26; p=NS, ρ Spearman=−0.499; FIG. 38E). Inagreement with the lack of increasing glycolysis, the expected increasein lactate levels (typical of sepsis) was not found but rather a slightdecrease (FIG. 38F and Tables 24A and 24B; p≤0.0255). The apparentinability of PBMCs from septic mice to shift from damaged mitochondrialrespiration to the glycolysis pathway reflects their disease severityand their failure to meet the energy demands of the immune system.

Taken together, these results show that in this CLP model of severesepsis, the majority of significantly altered parameters of organdysfunction strongly correlated with the MSS clinical score. Thesemarkers cover five of the main systems and organs that are damaged insepsis. Furthermore, based on the large area under the curves (AUC) oftheir ROCs, all markers that had a strong correlation with the clinicalscore can be used for prognosis in severe sepsis (Tables 24A and 24B;AUC >0.840). Therefore, the MSS clinical scoring system stronglyreflects the pathophysiological status of the mice, and as such can beused to evaluate the efficacy of our Allocetra-OTS treatment.

Allocetra-OTS Treatments

Adding Allocetra-OTS to Ertapenem, Dramatically Increased the Survivalof CLP Mice.

Since apoptotic cells were shown to bring an exaggeratedcytokine/chemokine response back to homeostasis, treating CLP-inducedseptic mice with Allocetra-OTS was envisioned to try rebalancing theimmune response as a potential therapy for sepsis. Mice underwent CLPprocedures to induce sepsis, as detailed in Methods above. Perioperativesurvival of mice from the CLP procedure using the isoflurane anesthesiamachine was considered high; only 3 out of 54 mice (5.5%) died duringthe first 24 h after the procedure (interval of 6.5-20 h) and wereexcluded from the study. 15 of 16 mice (94%) in the control group (CLPmice with vehicle injection-only) died of sepsis 24-72 hours after CLP.Compared to the CLP control group, ertapenem treatment with vehiclecontrol (n=15) had no significant effect on mouse survival, with only aslightly higher median survival (P>0.99; 31 h and 48 h, respectively),and similar mortality of 93%.

Initial Results

Shown in FIG. 33A, antibiotic treatment showed a non-significanttendency to ameliorate mortality of the mice (CLP+ Ertapenem+vehicle,n=15) compared to the control group (CLP only, n=16). Treating CLP micewith the combination of antibiotics and Allocetra-OTS significantlydelayed mortality and even prevented mortality in 60% of the animals(CLP+Ertapenem+Allocetra-OTS, n=20, p<0.001). In this model 90-100% ofmice die of sepsis within 50 hours. In comparison to the control group,the treated group reflected an approximately 10-fold improvement in thesurvival rate (p<0.001 in a log-rank analysis). As shown in FIG. 33B,Allocetra-OTS-treated mice had significantly lower clinical scoresindicating superior clinical condition.

Finally, the clinical score to serum cytokines/chemokines was correlatedwith in vivo measurements early on, and as shown in FIG. 33C,Allocetra-OTS downregulated pro-inflammatory cytokines/chemokinesrelated to monocyte/macrophage and dendritic cells activation. In thepreclinical study, Allocetra-OTS delayed and prevented mortality inanimal models with sepsis by reducing pro-inflammatorycytokines/chemokines and resetting the sepsis-related excessive immuneresponse following the initial immune response.

In order to determine the exact dosage that should be utilized, theAllocetra-OTS dose dependency in the CLP model was examined. As shown inFIG. 33D, the Allocetra-OTS effect was clear using 1×10⁶ cells and 3×10⁶cells. However, at 6×10⁶ cells it became indistinguishable from 10 and20×10⁶ cells, indicating that the minimum dose that should be used isbetween 1-6×10⁶ cells per 25 grams mouse. FIG. 33E supports thesefindings presenting additional dosages.

A single dose of 1-6×10⁶ Allocetra-OTS per 25-gram mouse, is equivalentto a dose of 40-240×10⁶. cells/kg cells in humans. In a previous humanstudy that focused on preventing complications post bone-marrowtransplantations, it was determined that 70×10⁶ apoptotic cells/kg weresufficient to cause a partial effect in humans and 140-210×10⁶ apoptoticcells/kg caused a significant effect (Mevorach, D., T. Zuckerman, I.Reiner, A. Shimoni, S. Samuel, A. Nagler, J. M. Rowe, and R. Or. 2014.Single infusion of donor mononuclear early apoptotic cells asprophylaxis for graft-versus-host disease in myeloablative HLA-matchedallogeneic bone marrow transplantation: a phase I/IIa clinical trial.Biol Blood Marrow Transplant 20:58-65). Therefore, it was concluded thatone and two dosages of 140×10⁶ cells/kg should be examined, where thehighest dosage would be 280×10⁶/kg (in two equal doses of 140×10⁶/kg).

TABLE 27 Spreadsheet for monitoring weight, survival, and clinical scoreof mice analyzed CLP (cecal CLP + CLP + CLP + CLP + CLP + ligationErtapenem + Ertapenem + Ertapenem + Ertapenem + Ertapenem + andAllocetra- Allocetra- Allocetra- Allocetra- Allocetra- puncture) OTS-OTS- OTS OTS OTS- only 1 × 10⁶ 3 × 10⁶ 6 × 10⁶ 10 × 10⁶ 20 × 10⁶ A B C BC D Hours n = 6 n = 8 n = 7 n = 8 n = 8 n = 8 0 100 100 100 100 100 1010.999 100 100 100 100 100 101 7 100 100 100 100 100 101 7.999 100 100100 100 100 101 10 100 100 100 100 100 101 23 100 100 100 100 100 10123.999 100 100 100 100 100 101 24 100 100 100 100 100 101 24.999 100 100100 100 100 101 25 100 100 100 100 100 101 26.999 100 100 100 100 100101 27 100 100 100 100 87.5 101 27.999 100 100 100 100 87.5 76 28 100100 100 100 75 76 28.999 100 100 100 100 75 76 29 66.6 87.5 100 87.5 7576 29.999 66.6 87.5 100 87.5 75 76 30 50 87.5 100 87.5 75 76 47.499 5087.5 100 87.5 75 76 47.5 33.3 87.5 71.4 87.5 75 76 48.999 33.3 87.5 71.487.5 75 76 49 16.6 87.5 71.4 87.5 75 76 50 16.6 87.5 71.4 87.5 75 7653.499 16.6 87.5 71.4 87.5 75 76 53.5 16.6 62.5 57.1 87.5 75 76 55.99916.6 62.5 57.1 87.5 75 76 56 16.6 62.5 42.8 87.5 75 76 72.4999 16.6 62.542.8 87.5 75 76 72.5 0 62.5 42.8 87.5 75 76 122 62.5 42.8 87.5 75 76 17762.5 42.8 87.5 75 76

The initial results showed that the recuperation rate of mice from theCLP procedure using the isoflurane anesthesia machine was very high.Only 3 out of 42 mice had died during the first night after theprocedure (interval of 6.5-20 h after procedure and were excluded fromthe study without 5 referring to different groups due to perioperativemortality).

Fifteen out of 16 mice of the Control group (CLP mice with vehicleinjection-only), died from sepsis 24-51 hours after CLP (94%). Ertapenemtreatment with vehicle control (n=15) slightly prolonged mice survivalto 30-97 h (p=NS), with the same survival rate: 14 out of 15 died(6.6%). Allocetra-OTS treatment combined with Ertapenem significantlyprolonged CLP-induced sepsis survival of mice (P<0.001, log-rank,10-fold increase in survival). Mice treated with Allocetra-OTS combinedwith Ertapenem died 29-146 h after CLP (n=20). Furthermore, 60% of micewere still alive in good condition, at day 7 (end of experiment,log-rank p value <0.001).

The cytokine/chemokines levels of typical mice from each group is shownin FIG. 33C.

In attempt to see dose-dependence, it was demonstrated that even 1 and 3million of Allocetra-OTS have an effect in severe sepsis.

Follow-Up Analysis

As described above, Allocetra-OTS treatment, combined with ertapenem,significantly prolonged the survival of the mice following CLP-inducedsepsis (FIG. 39A (As shown in FIG. 33A now with significance added);***P≤0.0005, log-rank test). Among the mice treated with Allocetra-OTSand ertapenem, eight of 20 (40%) died within 29-146 hours after CLP;however, the majority of the mice remained alive at the end of theexperiments 6-8 days post-CLP, and with significantly increased mediansurvival time of 160 h (FIG. 39B; P≤0.0074, Kruskal-Wallis nonparametricANOVA, multiple-comparisons adjusted with Dunn's test; 95% CI: 48 h-172h).

Allocetra-OTS Attenuates Sepsis Severity.

Murine survival had a strong reverse-correlation with clinical score(r-Pearson of −0.924; ***p<0.0001). Accordingly, treatment withAllocetra-OTS and ertapenem, substantially attenuated the appearance ofclinical symptoms. The final clinical score of Allocetra-OTS-treatedmice was significantly lower than that of the CLP control group and thatmice treated with Ertapenem alone (FIG. 39C; MSS plateau values of 7.78,14.81, and 14.37, respectively; ***p<0.0001, ordinary one-way ANOVA).

A dose-dependent, beneficial effect of Allocetra-OTS, was also observed;CLP mice that were treated with Allocetra-OTS doses between 1×10⁶ and20×10⁶ cells per mouse survived 42.85%-87.5% longer than vehicle-treatedCLP mice (FIG. 39D (as shown above in FIG. 33E now with significance;*P≤0.0115, log-rank test). Although even low doses of 1 and 3 millionAllocetra-OTS cells per mouse had a clear effect in severe sepsis,robust effects were seen only when using 6 million Allocetra-OTS cellsor more. Doses of 3-6 million Allocetra-OTS cells were not examined.

Allocetra-OTS Effects on Sepsis Severity are Achieved by Rebalancing theImmune-Response.

Previously, it was suggested that the dramatic effect of a singleapoptotic cell infusion on sepsis progression in the CLP model wasattributed rebalancing of the immune systems via interaction withmonocytes, macrophages, and dendritic cells Trahtemberg, U., and D.Mevorach. 2017. Apoptotic cells induced signaling for immune homeostasisin macrophages and dendritic cells. Front Immunol 8:1356). To examinethis concept, a wide panel of serum cytokines/chemokines was testedfollowing CLP, using the Luminex Multiplex kit (Millipore, Waltham,Mass., USA). As summarized in Table 28, 33 different cytokine andchemokine levels were elevated 24 h post-CLP in CLP mice compared tonaïve C57BL/6 mice.

TABLE 28 24-hour serum cytokine/chemokine change in mice afterCLP-induced sepsis (N = 6) compared to naïve mice (N = 2) Analyte CLPcompared to naïve mice CRP ↑↑ ENA-78 ↑↑ Eotaxin ↑↑ G-CSF ↑↑↑ GM-CSF ↑↑Gro-α ↑↑ IL-1α ↑↑ IL-1β ↑↑↑ IL-2 ↑↑ IL-2R ↑↑ IL-5 ↑↑ IL-6 ↑↑↑ IL-9 ↑↑IL-10 ↑↑↑ IL-12p70 ↑↑ IL-17A ↑↑↑ IL-18 ↑↑ IL-22 ↑↑↑ IL-23 ↑↑↑ IL-27 ↑↑↑IL-28 ↑↑ IL-31 ↑↑ IFNγ ↑↑ IP-10 ↑↑↑ LIF ↑↑↑ MCP-1 ↑↑ MCP-3 ↑↑ MIP-1α ↑↑MIP-1β ↑↑ MIP-2 ↑↑↑ RANTES ↑↑↑ TNFα ↑↑ VEGF-A ↑↑

Interestingly and unexpectedly, while treatment with ertapenemantibiotics alone had no beneficial effects on cytokine/chemokinelevels, combined treatment with ertapenem and Allocetra-OTS attenuatedand even abolished cytokine/chemokine release at 24 h and even 48 hafter sepsis induction (FIGS. 40A-40L). Reduced cytokine/chemokinerelease was observed for both pro-inflammatory and anti-inflammatorycytokines/chemokines. The cytokine storm rebalancing effect ofAllocetra-OTS corresponded well to the effect treatment with ofAllpcetra-OTS plus ertapenem on murine survival and sepsis severity.These findings strongly suggest that Allocetra-OTS confers its effectsthrough breaking the exaggerated cytokine storm that occurs in sepsisand rebalancing the immune response.

Discussion and Conclusions:

Triggering the innate immune system assures a common response pattern,regulated by the level of and variation in the repertoire of PAMPs andDAMPs, and the resulting signaling pathways that are activated. Thecomplementary nature of the pathways explains the overlapping yet uniqueearly inflammatory response to common Gram-negative bacteria,Gram-positive bacteria, fungal, and viral infections, as well as tissueinjury.

Sepsis is generally initiated by simultaneous recognition of eitherPAMPs or DAMPs by complement, toll-like receptors, NOD-like receptors,RIG-like receptors, mannose-binding lectin, and scavenger receptors.Recognition induces a complex intracellular signaling system withredundant and complementary activities, and activation of these multiplesignaling pathways ultimately leads to the expression of several commonclasses of genes that are involved in inflammation, adaptive immunity,and cellular metabolism. Apoptotic cells were shown to have a beneficialeffect on cytokine storms with downregulation of both anti- andpro-inflammatory cytokines derived from PAMPs and DAMPs, both in animalmodels and in in vitro models (See for example, Examples 2-8 above).Clearance of apoptotic cells allows immune homeostasis, generally leadsto a non-inflammatory state for both macrophages and dendritic cells(DCs) and contributes to the maintenance of peripheral homeostasis ofalmost all immune-triggered mechanisms in sepsis.

In this study of sepsis, a CLP-induced murine model for sepsis was usedthat successfully emulated human sepsis. This model simulated severesepsis with acute multiple organ dysfunction. Importantly, the MSSClinical Score method, adopted from Shrum et al (Shrum et al., 2014,ibid), was used to assess disease severity in tandem with multiple organanalysis, and the correlation between parameters of organ dysfunctionand disease severity was assessed. Indeed, CLP mice had low bloodpressure, poor cardiac output and lung dysfunction, as well as AKI, AKL,and thrombocytopenia that correlated with their clinical score. Thesecardiovascular and pulmonary failures are well documented in patientswith severe sepsis and septic shock, and in murine sepsis models.

The complement system mediates the activation of the innate immuneresponse against bacterial infection. However, this system isexcessively activated in sepsis, which may lead to deleterious effects.Accordingly, high levels of the complement proteins C3a and C5a weredetected in sepsis patients. While excessive generation of C5a causesharmful effects such as impaired neutrophil function andhyper-inflammation, some cohort studies of sepsis patients showed linksbetween higher C3a levels and survival or C3 depletion and highmortality. The opposing protective effects of C3 and the harmful effectsof C5 have been well-demonstrated in murine sepsis models of C3^(−/−)and C5^(−/−) or C3aR^(−/−) and C5aR^(−/−) after CLP; in these studies,C3-deficient mice had the poorest survival in comparison to WT andC5-deficient animals. Furthermore, C5aR^(−/−) mice were resistant toGram-negative bacteremia while C3aR^(−/−) were much more sensitive tothis infection.

Interestingly and surprisingly, the results of this study support thoseopposing effects, whereas it seems that the increased levels of C5a,together with decreased C3a levels corresponded to the high severity ofsepsis in our model. An explanation for this phenomenon may be in thefact that neutrophils have only C5a receptor whereas macrophages haveboth C5aR and C3aR.

The pathogenesis of sepsis is strongly related to vast changes inmetabolic homeostasis. Respiratory and cardiovascular dysfunction aswell as malnutrition lead to energetic crisis, while the hyperactivationof the immune system greatly raises energy demands. Recent studies haveprovided evidence for metabolic switching from oxidative phosphorylationto aerobic glycolysis to meet the increasing energy demands of activatedleukocytes in inflammation and sepsis. Accordingly, lactate, which isthe main by-product of aerobic glycolysis, was found to be greatlyincreased in both sepsis patients and in vivo animal models of sepsis.In contrary to most sepsis patients, who are diagnosed withhyperglycemia, mice in the CLP-based sepsis models present hypoglycemia,as also observed by here. It was speculated that this hypoglycemia wasthe consequence of rapid glucose catabolism for aerobic glycolysis byimmune cells. This was supported by the pro-inflammatory cytokineprofile following CLP; however, although a significant reduction of pHwas seen, it was surprisingly not accompanied by the expectedhyperlactatemia. Closer examination of mitochondrial respiratoryfunction and glycolytic capacity of PBMCs revealed heavily damagedmitochondria in CLP mice. The defects in energy production by oxidativephosphorylation were very severe and could not be rescued by thealternative glycolysis pathway. The inability to accelerate glycolysisas well as the apparent hypoglycemia, together with marked liver failurein CLP mice could be explained by mitochondrial dysfunction and severelyimpaired gluconeogenesis.

Overall, the majority of the significantly altered markers of organdysfunction, covering five of the main systems and organs that aredamaged in sepsis, also strongly correlated with the MSS Clinical Score.This correlation enabled the use of the MSS Clinical Score system as asurrogate method that reflects organ function (or dysfunction) in ourCLP mice model.

Using this model, it has been shown here that, surprisingly, a singledose of Allocetra-OTS not only significantly increased murine survival,but did so in a dose-dependent manner. Furthermore, the combinedtreatment with ertapenem antibiotic and Allocetra-OTS significantlyattenuated disease severity by almost fifty percent. Furthermore, inpatients undergoing bone marrow transplantation who had an elevatedcytokine profile, Allocetra was shown to be a safe and efficient with aclear dose-dependent effect starting at 140×10⁶ cells/kg. This was thebasis for selecting this dose for sepsis patients in the current trial.

The properties of apoptotic cells enable the mechanisms that lead totheir successful use as a therapeutic modality in sepsis, where most ofthese mechanisms are activated, as well as in various autoimmunediseases, organ transplantation, and graft-versus-host disease (GvHD).All of these conditions are characterized by cytokine storm. Indeed, inthe present study, it has been clearly shown that the beneficial effectsof Allocetra-OTS are achieved via its ability to interact with andrebalance the immune system.

Use of early apoptotic cells in the treatment of sepsis provided adramatic, unexpected effect on survival in severe sepsis. Furthermore,sepsis death was not only delayed but prevented. This surprising effectwas observed even with a dose of 1 million cells. Moreover, the effecton pro- and anti-inflammatory cytokines chemokines was unexpected as aneffect was expected only for proinflammatory cytokines. Such an effecton very high spectrum of cytokines chemokines was unknow before thisstudy.

The resemblance between the murine model and human sepsis, and theability to monitor sepsis severity as a derivative of organ dysfunction,as well as the initial promising positive effects of Allocetra-OTS,provide a valuable research tool for sepsis therapy. These results maylay the path for future use of Allocetra-OTS as the first effectivetherapy for sepsis.

It should be emphasized that treatment aiming to modulate the immuneresponse is not administered instead of antibiotic treatment, fluidresuscitation, and vasopressors. Rather apoptotic cells are an adjuvantand complementary treatment that rebalances the immune response.

Example 18: In Vivo Phase II Study of Sepsis

Objective:

After completing all pre-clinical safety and efficacy testing in animalsnoted above in Example 17, a randomized, multi-center,vehicle-controlled, comparative, open-label, study evaluating safety andefficacy of Allocetra-OTS for the prevention of organ dysfunction inpatients with sepsis will be performed.

The primary objective will be to evaluate the safety of Allocetra-OTS inpatients with sepsis. Secondary Objectives will be to assess preliminaryclinical efficacy and to support the proposed mechanism of action andbiological effect. Exploratory objectives will be to further explore thepotential mechanisms of action and biological effect.

Each patient will be followed for a period of 28 days, which is theaccepted standard for sepsis studies.

Methods:

This study is planned to be conducted in three clinical sites. The studyincludes three study groups (n=42 for all groups) that will be enrolledinto the study, two groups will be treated with one or two Allocetra-OTSdoses, and one group will be treated with vehicle (control group).Patients in each arm will be treated using the institutional standard ofcare (SOC) for sepsis.

Results:

It is expected that successful treatment of sepsis in humans will followthe trajectory provided in the pre-clinical trials in mice, whereinthere is an increase in survival, an anti-inflammatory effect oncytokines, and a reduction in organ damage or failure.

Example 19: Phase I Trial: Prevention of Sepsis-Related OrganDysfunction with Allocetra-OTS (P-SOFA-1)—Interim Data

Objectives:

Primary Objective:

To evaluate the safety of one dose and two doses of Allocetra-OTS inpatients with sepsis.

Secondary Objectives:

To assess preliminary clinical efficacy and to support the proposedmechanism of action and biological effect.

Exploratory Objective:

To explore possible additional mechanisms of action.

Methods:

Study Design:

Open label of one and two Allocetra-OTS doses in patients with sepsis.(See below for Allocetra-OTS preparation of dosage) Six (6) eligiblepatients were identified and recruited in the Emergency Room (ER) asthey were scheduled to be admitted to Intensive Care Unit (ICU) orIntermediate Unit (IMU). Patients were followed for safety and efficacyassessments for 28 days following Investigational Product (IP)administration.

The first three (3) patients were administered one dose of Allocetra-OTSby Intravenous (IV) infusion 140×10⁶±20% Allocetra-OTS cells/kg within24±6 hours following the time of meeting sepsis criteria—See below (time0).

Following 14 days (inclusive), an additional three patients wererecruited and received one IV dose of 140×10⁶±20% cell/kg ofAllocetra-OTS, as described above.

Following 14 days (inclusive), follow-up data of the additional threepatients (patients 4-6) will be submitted to the DSMB. DSMB evaluationsand recommendations will be submitted to the MoH. Following MoH approvaland notification to the EC, additional four (4) patients will berecruited. These four (4) patients (patients 7-10) will be administeredwith two doses of 140×10⁶±20% Allocetra-OTS cells/kg; the first within24±6 hours following diagnosis of sepsis, and the second 48±6 hoursfollowing the first treatment.

All study patients were treated using the institutional Standard of Care(SOC) for sepsis based on the Surviving Sepsis Campaign (Levy et al.(2018) The Surviving Sepsis Campaign Bundle: 2018 Update. Crit CareMed., 46(6):997-1000; Rhodes et al. (2017) Surviving Sepsis Campaign:International Guidelines for Management of Sepsis and Septic Shock:2016. Intensive Care Med. 2017 March; 43(3):304-377).

Study Procedures

The study included pre-screening, screening, treatment and follow-upperiods.

Pre-Screening period

Based on standard local ER procedures, patients were identified asmeeting clinical criteria for sepsis and start sepsis SOC. The time ofsepsis diagnosis was recorded as time 0.

Screening and Eligibility Confirmation Period (Time 0 to 24±6 hours)

Only patients with verbal Glasgow Coma Score (GCS) of 5 and totalGCS >13 were asked to participate in the study. Following patientsigning of the Inform Consent Form (ICF), screening procedures wereperformed. Screening procedures included collection of demographic data,complete and disease-related medical history and concomitantmedications, blood tests (biochemistry; hematology and coagulation),urinalysis, vital signs (Systolic and Diastolic Blood Pressures (BP),Respiratory Rate (RR), Temperature (T), Heart Rate (HR) and OxygenSaturation), physical examination, 12-lead ECG, infectious diseasescreening, echocardiogram (if indicated), pregnancy tests (for woman ofchildbearing potential), as well as qSOFA and SOFA scores calculations.Procedures that were conducted in the ER prior to the patient signingthe consent form as part of standard care were accepted. Followingscreening procedures and verification of inclusion and exclusioncriteria, eligibility was confirmed.

In addition to screening procedures, baseline tests were performed andincluded urine albumin/creatinine ratio, APACHE II score calculation,donor-specific antibodies, autoimmune serology and blood samples forpro- and anti-inflammatory cytokines, cell phenotype, transcriptionaldata in leukocytes, immune functions and apoptosis, metabolomics inserum and leukocytes, cell free DNA and endocrine parameters.

Treatment Period (Day 1 for patients that were treated with one dose andDay 1 to 3 for patients that will be treated with two doses):

Prior to investigational product (IP; Allocetra-OTS) treatment, patientsmust have been admitted to either the ICU or IMU and treated with SOCfor sepsis.

Premedication: patients received premedication prior to anyinvestigational product (IP; Allocetra-OTS) infusion, as follows:

-   -   40 mg methylprednisolone as IV injection—5±4 hours prior to        Allocetra-OTS transfusion.    -   12.5 mg promethazine as IV injection—30±30 minutes prior to        investigational product (IP; Allocetra-OTS) administration.

Patients treated with one dose (Patients 1-6): Within 24±6 hoursfollowing meeting sepsis criteria (time 0) patients receivedpremedication as indicated above, followed by one dose of Allocetra-OTSby IV infusion, using a volumetric pump, at a maximal rate of 102mL/hour, of 140×10⁶±20% cell/kg of Allocetra-OTS, in 375 ml RingerLactate solution.

Treatment day was designated as day 1. During investigational product(IP; Allocetra-OTS) infusion and the following 24 hours, procedures willbe conducted as follows:

-   -   Vital signs (Systolic and Diastolic Blood Pressures (BP),        Respiratory Rate (RR), Temperature (T), Heart Rate (HR) and        Oxygen Saturation) measurements were made—every 15±10 min during        the first hour of infusion, every 30±15 min from the second hour        till completion of investigational product (IP; Allocetra-OTS)        infusion, and every 6±3 hours following infusion completion.

Biochemistry and hematology blood tests at 60±60 minutes prior toinvestigational product (IP; Allocetra-OTS) administration.

Blood samples for pro- and anti-inflammatory cytokines, cell phenotype,transcriptional data in leukocytes, immune functions and apoptosis,metabolomics in serum and leukocytes, cell free DNA, endocrinology paneland ELISpot will be collected at 60±60 minutes prior to investigationalproduct (IP; Allocetra-OTS) administration.

SOFA and GCS calculation-were made 60±60 minutes Prior toinvestigational product (IP; Allocetra-OTS) administration.

Patients treated with two doses (Patients 7-10): Within 24±6 hoursfollowing sepsis diagnosis (time 0) patients will receive premedicationas indicated above, followed by the first dose of IV infusion, and after48±6 hours from the first treatment will receive premedication asindicated above, followed by the second dose of IV infusion. Allinfusion will be administered using a volumetric pump at a maximal rateof 102 mL/hour, of 140×10⁶±20% cell/kg of Allocetra-OTS, in 375 mlRinger Lactate solution.

Treatment day of the first dose will be designated as day 1, andtreatment day of the second dose will be designated as day 3. During thetreatment period (days 1 to 3) procedures will be conducted as follows:

-   -   Vital signs (Systolic and Diastolic Blood Pressures (BP),        Respiratory Rate (RR), Temperature (T), Heart Rate (HR) and        Oxygen Saturation) measurements as follows: on days 1 and 3:        every 15±10 min during the first hour of infusion, every 30±15        min until completion of investigational product (IP;        Allocetra-OTS) infusion, and every 6±3 hours following infusion        completion; on day 2 every 6±3 hours.

Blood tests (biochemistry and hematology):

-   -   Day 1: 60±60 minutes prior to investigational product (IP;        Allocetra-OTS) administration.    -   Day 2: 24±6 hours following completion of first investigational        product (IP; Allocetra-OTS) infusion.    -   Day 3: 48±6 hours following completion of first investigational        product (IP; Allocetra-OTS) infusion and prior to second        investigational product (IP; Allocetra-OTS) infusion.

Blood samples for pro- and anti-inflammatory cytokines, cell phenotype,transcriptional data in leukocytes, immune functions and apoptosis,metabolomics in serum and leukocytes and cell free DNA, as follows:

-   -   Day 1: 60±60 minutes prior to first investigational product (IP;        Allocetra-OTS) administration.    -   Day 2: 24±6 hours following completion of first investigational        product (IP; Allocetra-OTS) infusion.    -   Day 3: 60±60 minutes prior to second investigational product        (IP; Allocetra-OTS) administration.

ELISpot on day 1—60±60 minutes prior to first investigational product(IP; Allocetra-OTS) administration.

12-lead ECG: on day 2, 24±2 hours following completion of the firstinvestigational product (IP; Allocetra-OTS) infusion.

SOFA and Glasgow coma scale (GCS) scores calculation: on days 1, 2 and3.

Follow Up Period (Day 2 to 28 for the single-dose patients and Day 4 to28 for the two-dose patients):

Patients were followed-up to study day 28 following firstinvestigational product (IP; Allocetra-OTS) treatment for safety andefficacy evaluations as follows:

-   -   Adverse events and concomitant medication: continuously.    -   Vital signs (Systolic and Diastolic Blood Pressures (BP),        Respiratory Rate (RR), Temperature (T), Heart Rate (HR) and        Oxygen Saturation), as follows: for single dose—twice daily on        days 2 and 3 and once daily as long as the patient was        hospitalized. For two doses—twice daily on days 4 and 5 and once        daily as long as the patient is in hospitalized. If patient is        discharged, vital signs will be measured on day 7, 14 and 28        visits.    -   Blood tests (biochemistry and hematology) were taken once daily        until day 7 and on days 11±1, 14±1, 18±1, 21±1, 24±1 and 28±2 if        the patient was hospitalized. If patient was discharged, these        tests were measured on day 7±1, 14±2 and 28±2 visits.    -   Blood samples for pro- and anti-inflammatory cytokines, immune        functions and apoptosis, metabolomics in serum and leukocytes        and cell free DNA on days 2, 3, 4, 7±1, 14±2 and 28±2.    -   Blood samples for cell phenotype and transcriptional data in        leukocytes on days 2, 3, 4 and 28±2.    -   Blood samples for endocrinology panel on days 4±1 and 28±2.    -   12-lead ECG: on day 2 for patients treated with one dose and on        day 4 for patients treated with two doses (24±2 hours following        investigational product (IP; Allocetra-OTS) administration), as        well as on days 14±2 and 28±2 for all patients.    -   SOFA scores were documented on days 2, 3, 7, 14±2, 28±2.    -   Donor-specific antibodies, ELISpot and autoimmune serology will        be evaluated on day 28±2.

Study Duration:

For each participating patient, the duration in the study was 28 daysfrom investigational product (IP; Allocetra-OTS) treatment.

Study Population:

As of this time, the Study included 6 patients, with a goal of having atotal of 10 patients.

Inclusion/Exclusion Criteria:

Inclusion Criteria:

-   -   Suspected, presumed or documented infection from any source.    -   Initiation of antibiotics.    -   Meets Sepsis 3 criteria: The presence of organ dysfunction as        identified by a total SOFA score ≥2 points above baseline.    -   Adult male or female, age between 18 and 85.    -   GCS of >13 with verbal score of 5.    -   Signed written informed consent by the patient.

Exclusion Criteria:

-   -   Participation in an interventional investigational trial within        30 days prior to diagnosis of sepsis.    -   Significant trauma requiring hospitalization within 30 days        prior to diagnosis of sepsis.    -   Surgical intervention, or plan for surgical intervention, or        hospitalization within 30 days prior or after the scheduled        investigational product (IP; Allocetra-OTS) infusion.    -   Pregnancy or breast-feeding female.    -   Progressive or poorly controlled malignancies or <6 month after        active treatment for cancer (chemotherapy or irradiation).    -   Terminally ill patients defined as patients that prior to the        current hospitalization are expected to live <6 months (as        assessed by the physician responsible for the patient).    -   Known active acute or chronic viral infections, e.g. Hepatitis B        Virus (HBV) or Hepatitis C Virus (HCV), Human Immunodeficiency        Virus (HIV) or other chronic infection.    -   Known severe chronic respiratory health problems with severe        pulmonary hypertension (≥40 mmHg) or respirator dependency.    -   Known active upper gastrointestinal (GI) tract ulceration or        hepatic dysfunction including but not limited to: biopsy-proven        cirrhosis; portal hypertension; episodes of past upper GI        bleeding attributed to portal hypertension; or prior episodes of        hepatic failure, encephalopathy, or coma.    -   Known New York Heart Association (NYHA) class IV heart failure        or unstable angina, ventricular arrhythmias, active ischemic        heart disease, or myocardial infarction within six months prior        to diagnosis of sepsis.    -   Known immunocompromised state or medications known to be        immunosuppressive.    -   Organ allograft or previous history of stem cell        transplantation.

Allocetra-OTS Product, Route of Administration, and Dosage Form

Allocetra-OTS is a cell-based therapeutic composed of donor earlyapoptotic cells. The Allocetra-OTS product contained allogeneic donormononuclear enriched cells in the form of a liquid suspension with atleast 40% early apoptotic cells. Early apoptotic cells were prepared asper Example 1 above. The suspension was prepared with Ringer's lactatesolution and stored at 2-8° C. until 45±25 minutes before infusion andat room temperature thereafter. Allocetra-OTS contains 140×10⁶±20% cellsper kg of recipient body weight in a total volume of 375 mL in atransfer pack that underwent irradiation and was administrated using avolumetric pump, at a starting rate of 48 mL/hour (16 drops per minute)with a gradual increase every 15-25 minutes of 15 mL/hour (additional 5drops per minute) to a maximal rate of 102 mL/hour, as follows: 63 (21drops) mL/hour; 78 (26 drops) mL/hour; 93 (31 drops) mL/hour; 102 (34drops) mL/hour.

During investigational product (IP; Allocetra-OTS) administration noother I.V. fluids such as Ringer's lactate or Normal Saline were givenin parallel, unless medically indicated due to volume depletion.

The Allocetra-OTS was administered to the patient within 72 hours ofcompleting the manufacturing process.

Standard of Care (SOC)

The SOC was according to the Surviving Sepsis Campaign guidelines (Levy,M. M., L. E. Evans, and A. Rhodes. 2018. The surviving sepsis campaignbundle: 2018 update. Crit Care Med 46:997-1000; Rhodes, A., L. E. Evans,W. Alhazzani, M. M. Levy, M. Antonelli, R. Ferrer, A. Kumar, J. E.Sevransky, C. L. Sprung, M. E. Nunnally, B. Rochwerg, G. D. Rubenfeld,D. C. Angus, D. Annane, R. J. Beale, G. J. Bellinghan, G. R. Bernard, J.D. Chiche, C. Coopersmith, D. P. De Backer, C. J. French, S. Fujishima,H. Gerlach, J. L. Hidalgo, S. M. Hollenberg, A. E. Jones, D. R. Karnad,R. M. Kleinpell, Y. Koh, T. C. Lisboa, F. R. Machado, J. J. Marini, J.C. Marshall, J. E. Mazuski, L. A. McIntyre, A. S. McLean, S. Mehta, R.P. Moreno, J. Myburgh, P. Navalesi, O. Nishida, T. M. Osborn, A. Perner,C. M. Plunkett, M. Ranieri, C. A. Schorr, M. A. Seckel, C. W. Seymour,L. Shieh, K. A. Shukri, S. Q. Simpson, M. Singer, B. T. Thompson, S. R.Townsend, T. Van der Poll, J. L. Vincent, W. J. Wiersinga, J. L.Zimmerman, and R. P. Dellinger. 2017. Surviving sepsis campaign:international guidelines for management of sepsis and septic shock:2016. Intensive Care Med 43:304-377) with allowance for variance basedon institutional standards. Institutional SOC may include intravenous(IV) fluids including crystalloids with or without albumin, other bloodproducts according to accepted indications, vasopressors and inotropes,antibiotics, anti-viral or anti-fungal agents and corticosteroids ifindicated.

Premedication

Patients received 40 mg methylprednisolone and 12.5 mg of I.V.promethazine, as IV injections, prior to Allocetra-OTS transfusion.

Concomitant Medications

Prohibited medications: Significant immune suppressing agents includingchronic corticosteroids >10 mg/day, azathioprine, cyclosporine,cyclophosphamide, and any biological treatment.

Safety Endpoint (primary)

Assessment of safety was accomplished by determining the clinicaloutcome and the number of participants with any Adverse Events (AE),Serious Adverse Events (SAE) and fatal SAE.

Efficacy Endpoints/Outcome Measures (Secondary)

The following efficacy endpoints were measured throughout the studyperiod:

-   -   Any one of the following organ function or support measurements        recorded throughout the 28-days study period:        -   Ventilator-free days, and/or        -   Vasopressor-free days, and/or        -   Days without renal replacement therapy (dialysis) and/or            days with creatinine ≤baseline+20%, and/or        -   Days with ≥100×109/L platelets count, and/or        -   Days with ≤three times normal ALT and AST levels and/or ≤two            times normal bilirubin levels, and/or        -   Days with return to GCS 15.    -   Mortality from any cause.    -   Cumulative days in ICU or IMU and/or in hospital.    -   Time to CRP <20 mg/L.    -   Time to normal+20% lactate levels.

Exploratory Endpoints

The following exploratory biological tests were or will be measured atbaseline and at days 1, 2, 3, 4, 7, 14 and 28 post first investigationalproduct (IP; Allocetra-OTS) treatment:

-   -   Pro- and anti-inflammatory cytokines: MIP-1 beta, TNF alpha,        MCP-1, IL-1R, IL-1 beta, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7,        IL-8, IL-9, IL-15, MIP-1 alpha, IL-22 and GMCSF.    -   Immune functions and apoptosis: HMGB1, histone levels, and        complement.    -   Metabolomics in serum and leukocytes: Pyruvate, FFAs,        glycolysis, mitochondrial function, leptin, ghrelin and        glucagon.    -   Cell free DNA

The following exploratory blood test were or will be measured atbaseline and at days 1,2, 3 (prior investigational product (IP;Allocetra-OTS) treatment for patients treated with two doses), 4, and 28post first investigational product (IP; Allocetra-OTS) treatment:

-   -   Cell Phenotype: T regs, CD4, CD8, NK and B, monocytes, dendritic        cells and function and expressions of PD-1, PDL-1 and BTLA.    -   Transcriptional data in leukocytes.

ELISpot was or will be measured on baseline, day 1 and day 28.

-   -   Endocrinology panel at baseline and on days 1, 4 and 28:        Cortisol, ACTH, FT3, FT4, TSH, growth hormone and insulin.

The following exploratory blood tests was or will be measured atbaseline and on day 28:

-   -   Donor specific antibodies: Panel Reactive Antibodies (PRA).    -   Autoimmune serology panel: ANA, Anti-DNA, Anti-RNP, Anti-SSA,        Anti-SSB, Cardiolipin IgG and Cardiolipin IgM

Statistical Analysis

Data from all clinical assessments, whether explicitly referred to inthe statistics section or not, was and will be listed and, whereappropriate, summarized by dose group and by other categoricalinformation of interest using descriptive statistics.

Summary statistics (arithmetic mean, standard deviation, minimum value,lower quartile, median, upper quartile, maximum value, number ofnon-missing values) was and will be presented for continuous variables(absolute values at each time point and changes from baseline, ifappropriate) and counts and percentages will be presented forcategorical variables. Where appropriate, the presentation of resultsincludes shift tables, plots, statistical tests or confidence intervals.

Results:

The results presented in FIGS. 41A-41C through FIGS. 50A-50B providepositive interim efficacy data from an ongoing Phase Ib clinical trialin patients with severe sepsis. The interim analysis is based on adataset with a total of 43 patients with severe sepsis (6 treatedpatients and 37 historical controls), all hospitalized at HadassahMedical Center (FIGS. 41A-41C and 42 ). Six patients admitted tointensive care unit with sepsis were administered with off-the-shelfAllocetra (“OTS Allocetra”; early apoptotic cells as described above)upon their admission into the intensive care unit, while 37 patients arematched controls that have received standard of care during 2016-2019but did not received Allocetra treatment.

The primary safety parameter was ±mortality at 28 days. Surprisingly, noAllocetra-treated patients died (0/6 (0%)) compared with 11/37 (29%) inthe matched control group (FIGS. 43, 46A, and 46B). Efficacy parametersthat were analyzed include organ failure clinical SOFA score (the higherthe score, the negative the clinical condition of various organs) (FIGS.48A-48D, 49A-49B, and 50A-50B), as well as mortality (FIGS. 46A-46B),recovery from sepsis (FIGS. 44A-44B and FIG. 45 ), number of days ofhospitalization in the intensive care unit (FIG. 45 and FIGS. 47A-47B).

Table 29 below provides a summary of the interim results showing therobust safety and efficacy profile demonstrated by Allocetra-OTSadministration to patients with severe sepsis. The matching of the 37patients to the OTS Allocetra-treated group was based on similar organfailure clinical SOFA score at admission, overall clinical state, agegroup, sex, and source of severe sepsis (pneumonia, endovacular, orurinal tract infections). All matched patients were treated at the samehospital as the Allocetra-treated group. A summary of the matchingcharacteristics is presented in Table 30.

TABLE 29 Summary Interim Results Matched Treated with Sepsis OutcomesUntreated Group OTS Allocetra % of patients that recovered 48% 100% from Sepsis within 28 days % Mortality of patients 29% 0% Organ failureclinical SOFA Avg. at admission: Avg. at admission: score at admissionvs maximal 3.98 4.5 reached during hospitalization Organ failureclinical score at Avg. Maximal: Avg. Maximal: admission vs maximalreached 8.11 4.5 during hospitalization % of patients with organ Medianat Median at failure clinical score that admission: 4 admission: 4.5increased by 28 days % of patients with organ Median Maximal: 8 MedianMaximal: 45 failure SOFA clinical score that increased by 4 or moreduring 28 days % of patient mortality among 78% 0% those with organfailure clinical SOFA score that increased by 4 or more during 28 days %of patients still in intensive 57% 0% care unit after 6 days

TABLE 30 Summary Allocetra-OTS Patient and Matching ControlCharacteristics Matched Treated with Matching Characteristics UntreatedGroup OTS Allocetra Source of sepsis Pneumonia 67% 68% Endovascular ((MRSA) 16% 17% Urinal tract infection 17% 15% Age group distributionAvg. age: 69.2 Avg. age: 69.8 Median age: 69 Median age: 70.5 SOFA atadmission Avg. at admission: 3.98 Avg. at admission: 4.50 Median atMedian at admission: 4 admission: 4.50 Sex All male All male

Summary:

The interim results of the Phase Ib clinical trial demonstrate positiveinterim efficacy for safety and efficacy, in treating patients withsepsis using Allocetra-OTS (“Off the Shelf”) early apoptotic cells. Nomortalities were observed and time to recover from sepsis (time to SPFAscore<2) and hospital length of stay, were significantly reducedcompared to historical controls.

Example 20: Phase I Trial: Prevention of Sepsis-Related OrganDysfunction with Allocetra-OTS (P-SOFA-1)—Phase 1B Data Update toExample 19

Objectives:

The primary aim of this phase IB study was to determine the safetyprofile of apoptotic cell infusion (Allocetra-OTS) in subjectspresenting to the emergency room with sepsis. Sepsis was determined byclinical infections and Sequential Organ Failure Assessment (SOFA)scores >2. The secondary aims were to measure organ dysfunction,intensive care unit (ICU) and hospital stays, and mortality, that werecompared to historical controls. Exploratory endpoints includedmeasuring immune modulator agents to elucidate the mechanism of actionusing Luminex® analysis.

Methods:

Methods were as described in Example 19, wherein the additional patientswere treated, for example but not limited to the patient set treatedwith two doses of Allocetra. A skilled artisan would therefor understandthat wherein portions of the methods of Example 19 refer to work to beperformed, it is these methods that were performed and are reportedherein in Example 20.

Results:

Twelve patients were screened, and ten patients were included in thestudy. Patients 04 and 05 did not meet inclusion criteria. The DSMB metfollowing inclusion of three (patients 01-03) and six patients (01-03and patients 06-08) for a safety review and approval to continue thestudy, and for final review after 10 patients. Allocetra-OTS infusion inthe first three patients met the protocol for defined safety criteria atday 14 and the study proceeded to the second round of patientrecruitment (patients 06-08) at the same dose, which also met theprotocol-defined criteria for safety. The study then proceeded torecruit four additional patients (cohort 2, patients 09-12), whoreceived two doses of Allocetra-OTS.

Patient characteristics are presented in Table 31. All patients had aGlasgow Coma Score (GCS) of at least 13 (verbal 5/5) at enrollment dueto the requirement to obtain consent directly from the study subject. Atenrollment, the average APACHE II score was 12.3 (range 8-21) and theaverage SOFA score was 3.4 (range 2-6). All patients fulfilled inclusionand exclusion criteria and met the 2016 definition of sepsis. (Singer,M., C. S. Deutschman, C. W. Seymour, M. Shankar-Hari, D. Annane, M.Bauer, R. Bellomo, G. R. Bernard, J.-D. Chiche, C. M. Coopersmith, R. S.Hotchkiss, M. M. Levy, J. C. Marshall, G. S. Martin, S. M. Opal, G. D.Rubenfeld, T. van der Poll, J.-L. Vincent, and D. C. Angus. 2016. TheThird International Consensus definitions for sepsis and septic shock(Sepsis-3). JAMA 315:801-810) In this study, any source of infection wasincluded, and the patients presented with four types of infections;pneumonia (five patients), biliary tract infection (three patients),endovascular (one), and urinary tract infection (one) (Table 31).

Clinical course.

All study subjects fitted the definition of sepsis and inclusioncriteria and were hospitalized on admission in either the ICU or theIMU. All patients had at least two organ systems involved (range 2-5systems). Acute kidney injury (three patients), cardiovascularinvolvement (three patients), hepatic involvement (seven patients),hematological (eight patients) and pulmonary involvement (five patients)was seen in the treated patients before IP administration. All patientsrecovered from the septic condition and were discharged alive from thehospital following the resolution of sepsis and completion of 28 days offollow-up.

TABLE 31 Patient and matched-historical control characteristics Sepsissource: Sepsis source: Sepsis source: Biliary-Infection. UTI.Endovascular. Sepsis source: Sepsis source: Treated Patients TreatedPatients Treated Patients Total Sepsis Total Sepsis Pneumonia Pneumonia(n = 3, 30%)/ (1, 10)/ (1, 10%)/ Treated patients Matched-ControlsTreated Patients Matched Controls Matched controls Matched controlsMatched controls (n = 10, 100%) (n = 37, 100%) (n = 5, 50%) (n = 19,51.3%) (n = 10, 27.0%) (n = 5, 13/5%) (n = 3, 8.1%)) Mean age 71.9(51-83) N/A** (range) 67.8 (51-79) 67.8 (50-79) 76.3 (70-83)/ N/A**N/A** (range) 74.8 (67-83) Male:female 8/2 31/6 4/1 15/4 2:1/8:2 N/A**N/A** Predicted mortality Maximal Predicted SOFA based on SOFA scoremortality score at SOFA at following APACHE based on Patient Screeningpresentation* IP infusion II Score APACHE II # 28 days Outcome 01-001, 2 8% 2 12 15.9% Alive. Sepsis and organ dysfunction male resolvedPneumonia 01-002, 3 10% 3 9  9.9% Alive. Sepsis and organ dysfunctionmale resolved Pneumonia 01-003, 6 16% 6 15   21% Alive. Sepsis and organdysfunction male resolved Pneumonia 01-006, 3 10% 3 11 12.9% Alive.Sepsis and organ dysfunction male resolved Pneumonia 01-007, 6 16% 6 2138.9% Alive. Sepsis and organ dysfunction male resolved Endovascular01-008, 3 10% 3 9  9.9% Alive. Sepsis and organ dysfunction maleresolved UTI 01-009, 3 10% 3 13 18.6% Alive. Sepsis and organdysfunction male resolved Biliary 01-010, 5 13% 5 16 23.2 Alive. Sepsisand organ dysfunction male resolved Biliary 01-011, 2  8% 2 8  8.9%Alive. Sepsis and organ dysfunction woman resolved Biliary 01-012, 2  8%2 15   21% Alive. Sepsis and organ dysfunction woman resolved PneumoniaAverage 3.4 (2-6) 11.9 (8-16)% 3.4 (2-6) 12.3 (8-21) 18.0 (9.9-38.9)%Actual mortality: Treated patients: 0%; (range) Matched-historicalcontrols: 27% *(Raith, E. P., A. A. Udy, M. Bailey, S. McGloughlin, C.MacIsaac, R. Bellomo, D. V. Pilcher, Australian, O. New ZealandIntensive Care Society Centre for, and E. Resource. 2017. Prognosticaccuracy of the SOFA score, SIRS criteria, and qSOFA score forin-hospital mortality among adults with suspected infection admitted tothe intensive care unit. JAMA 317: 290-300.) # (Rowan, K. M., J. H.Kerr, E. Major, K. McPherson, A. Short, and M. P. Vessey. 1993.Intensive Care Society's APACHE II study in Britain and Ireland--I:Variations in case mix of adult admissions to general intensive careunits and impact on outcome. BMJ 307: 972-977.) **Not available at thistime.

Laboratory Results.

Laboratory evaluation included complete blood count (CBC), biochemistry,blood gases, C-reactive protein (CRP), and lactate. In 5/10 patients,elevated white blood cells (WBC) counts were evident in the first days,with gradual return to normal levels (FIG. 1 ). The behavior of the WBCcount did not correlate with the source of sepsis. (FIG. 51A)Neutrophilia was observed at admission in 6/10 patients and lymphopeniain 9/10 patients. (FIGS. 51B and 51C, respectively). Lymphopeniadeveloped in one patient (01) 1 day after admission, but beforeinvestigational product (IP) administration. The only patient withoutlymphopenia was patient 11, who presented with a biliary infection. Allpatients had a gradual increase in lymphocyte numbers and 6/9 (66%)recovered to normal levels whereas 3/9 (33%, patients 03, 06, 07) hadmoderate recovery levels. In summary, WBC changes were remarkable withelevated WBC and neutrophilia in 50-60% of patients and decreasedlymphocytes in 90%. By day 28, most patients recovered to within normalparameters.

C-reactive protein (CRP) declined in parallel with resolution ofinflammation (FIG. 51D). Two patients (07 and 12) had a slower declineof CRP. The first (07) was a patient on chronic dialysis presenting withan endovascular infection that needed antibiotics for 6 weeks, and thesecond (12) was a patient with pneumonia. Lactate levels were elevatedin 3/10 patients upon admission (01, 02, 08) and were normal or nearnormal in the following days. Three patients had higher lactate levelson day 28 (01, 03, 09) despite clinical and laboratory resolution ofsepsis, in the range of 3.4 to 6.5 mmol/L (normal value up to 2.2mmol/L).

Safety Parameters.

Safety was evaluated by serious adverse events (SAEs) and adverse events(AEs). All patients survived 28 days of follow-up. Of note, efficacyparameters (presented below), like survival, overlapped with additionalsafety parameters. There were no serious unexpected serious adversereactions (S USARs) and none of the subjects experienced an SAE, severeor moderate AE, or discontinued the study due to an AE. Nearly allsubjects (9/10; 90%) experienced at least 1 AE and in average 4.4 AEs;The most common AEs were associated with laboratory investigations(Table 32) and all were mild in intensity and most were unrelated to IP.The six possibly related AEs were transiently elevated temperature, mildtransient tachycardia, transient hypoglycemia, and transient dizziness,each in one patient, and rigor episodes in two patients receiving onedose. All these AEs could have been related to the septic condition.However, since the two episodes of rigor occurred in patients receivinga high fusion rate (>150 mL/h), the last 8 IP administrations were givenat a slower rate (up to >=108 mL/hr) with no rigors documented.Autoantibody induction, including ANA, anticardiolipin, and anti-DNAwere examined during the study period and found to be negative.

TABLE 32 Complete list of Adverse events. Patient Action number/Relationship Taken with Gender/ Severity/ to study Study Outcome of NoAge Adverse Event Intensity drug Treatment Adverse Event 1 01/M/67Rigors Not Possibly Drug Recovered/ serious/ related interrupted;Resolved Mild rate changed. dose unchanged 2 02/M/51 Tachycardia NotPossibly Dose not Recovered/ serious/ related changed Resolved Mild 302/M/51 Temperature Not Possibly Dose not Recovered/ elevation to 37.9serious/ related changed Resolved C. Mild 4 03M/74 Elevated liver NotNot related Not Recovered/ enzymes serious/ applicable Resolved Mild 507/M/65 Dizziness Not Possibly Dose not Recovered/ serious/ relatedchanged Resolved Mild 6 07/M/65 Anemia Not Not related Not Not serious/applicable Recovered/Not Mild Resolved 7 07/M/65 Gamma- Not Not relatedNot Not glutamyl serious/ applicable Recovered/Not transferase out MildResolved of range - high 8 07/M/65 Lactic Not Not related Not Recovered/dehydrogenase serious/ applicable Resolved out of range - Mild high 907M/65 Creatine Not Not related Not Recovered/ phosphokinase serious/applicable Resolved out of range - Mild high 10 07/M/65 Alkaline Not Notrelated Not Not phosphatase out serious/ applicable Recovered/Not ofrange - high Mild Resolved 11 07/M/65 WBC out of Not Not related NotRecovered/ range - high serious/ applicable Resolved Mild 12 07/M/65Neutrophils out Nor Not related Not Recovered/ of range - high serious/applicable Resolved Mild 13 07/M/65 Lactic Not Not related NotRecovered/ dehydrogenase serious/ applicable Resolved out of range -Mild high 14 08/M/82 Rigors Not Possibly Not Recovered/ serious/ relatedapplicable Resolved Mild 15 08/M/82 Glucose out of Not Not related NotRecovered/ range serious/ applicable Resolved Mild 16 08/M/82 Sodium outof Not Not related Not Recovered/ range serious/ applicable ResolvedMild 17 08/M/82 General atopy Not Not related Not Recovered/ serious/applicable Resolved Mild 18 08/M/82 Lymphocytes Not Not related NotRecovered/ out of normal serious/ applicable Resolved range - high Mild19 08/M/82 Lactic Not Not related Not Recovered/ dehydrogenase serious/applicable Resolved out of range - Mild high 20 08/M/82 Bilirubin out ofNot Not related Not Recovered/ range serious/ applicable Resolved Mild21 08/M/82 Glucose out of Not Not related Not Recovered/ range serious/applicable Resolved Mild 22 09/M/83 Elevated ALK.P Not Not related NotRecovered/ serious/ applicable Resolved Mild 23 09/M/83 Elevated LDH NotNot related Not Recovered/ serious/ applicable Resolved Mild 24 09/M/83Elevated WBC Not Not related Not Recovered/ serious/ applicable ResolvedMild 25 09/M/83 Lymphocyte Not Not related Not Recovered/ ABS decreaseserious/ applicable Resolved Mild 26 09/M/83 Low glucose Not PossiblyNot Not level serious/ related applicable Recovered/Not Mild Resolved 2710/M/70 Diarrhea Not Unlikely Dose not Recovered/ serious/ relatedchanged Resolved Mild 28 11/F/76 Low total No Mild Not related Dose notchanged protein blood level 29 11/F/76 Low albumin No Mild Not relatedDose not changed blood level 30 11/F/76 Elevated LDH No Mild Not relatedNot applicable blood level 31 11/F/76 Low CPK blood No Mild Not relatedNot applicable level 32 11/F/76 LowpPotassium No Mild Not related Dosenot changed blood level 33 11/F/76 Elevated CRP No Mild Not related Dosenot changed blood level 34 11/F/76 Elevated lactate No Mild Not relatedNot applicable blood level 35 11/F/76 Elevated No Mild Not related Notapplicable alkaline phosphotase blood level 36 12/F/68 Elevated lactateNo Mild Not related Not applicable blood level 37 12/F/68 Low total NoMild Not related Not applicable bilirubine blood level 38 12/F/68Elevated ALT No Mild Not related Not applicable blood level 39 12/F/68Low ALT blood No Mild Not related Not applicable level 40 12/F/68Elevated AST No Mild Not related Not applicable blood level 41 12/F/68Elevated LDH No Mild Not related Not applicable blood level 42 12/F/68Elevated No Mild Not related Not applicable alkaline phosphotase bloodlevel 43 12/F/68 Low BUN No Mild Not related Not applicable blood level44 12/F/68 Elevated lactate No Mild Not related Not applicable bloodlevel

Autoimmunity and Autoantibodies.

Autoantibodies and autoimmunity were evaluated before and on day 28since in animal studies injecting late apoptotic/necrotic cells inducedtransient autoantibodies. No autoimmunity developed in any of the studysubjects during the study period. The antinuclear antibody screen (ANA),which is considered a major screening tool for autoantibodies, wasnegative at presentation in 8/10 patients and remained negative in alleight at day 28. In two patients it was low-positive before IPadministration; it remained low-positive in one and disappeared in thesecond. IgM and IgG anticardiolipin were negative in 10/10 patients atscreening and remained negative in all.

Anti-DNA was not examined at initial screening, due to negative ANA inmost patients, but was negative in all patients examined at day 28.Anti-SSA/SSB/RNP/Sm were examined mainly on day 28 and in some patientsat initial screening. Only one patient (08) had very low positive RNP(20.61 vs 20, which is defined as negative) on day 28. This wasconsidered non-significant.

In conclusion, after administration of 14 IV doses of Allocetra-OTS in10 patients, there was no evidence of autoimmunity or autoantibodies.

Anti-HLA Antibodies.

Anti-donor HLA antibodies were examined in the first 6 patients on day28 following one dose administration and no anti-HLA antibody was found.Additional results from 2 doses are pending.

Preliminary Efficacy Results:

mortality. No deaths occurred among the 10 study subjects. The expectednumber of deaths in hospitalized septic patients ranges between 30% and45%; (Finfer, S., R. Bellomo, J. Lipman, C. French, G. Dobb, and J.Myburgh. 2004. Adult-population incidence of severe sepsis in Australianand New Zealand intensive care units. Intensive Care Med 30:589-596;Fleischmann, C., A. Scherag, N. K. Adhikari, C. S. Hartog, T. Tsaganos,P. Schlattmann, D. C. Angus, and K. Reinhart. 2016. Assessment of globalincidence and mortality of hospital-treated depsis. Current estimatesand limitations. Am J Respir Crit Care Med 193:259-272; Machado, F. R.,A. B. Cavalcanti, F. A. Bozza, E. M. Ferreira, F. S. Angotti Carrara, J.L. Sousa, N. Caixeta, R. Salomao, D. C. Angus, and L. C. Pontes Azevedo.2017. The epidemiology of sepsis in Brazilian intensive care units (theSepsis PREvalence Assessment Database, SPREAD): an observational study.Lancet Infect Dis 17:1180-1189; Reinhart, K., R. Daniels, N. Kissoon, F.R. Machado, R. D. Schachter, and S. Finfer. 2017. Recognizing sepsis asa global health priority—a WHO resolution. N Engl J Med 377:414-417;Rhee, C., R. Dantes, L. Epstein, D. J. Murphy, C. W. Seymour, T. J.Iwashyna, S. S. Kadri, D. C. Angus, R. L. Danner, A. E. Fiore, J. A.Jernigan, G. S. Martin, E. Septimus, D. K. Warren, A. Karcz, C. Chan, J.T. Menchaca, R. Wang, S. Gruber, and M. Klompas. 2017. Incidence andtrends of sepsis in US hospitals using clinical vs claims data,2009-2014. JAMA 318:1241-1249) however, in the specific studiedpopulation, with a GCS of at least 13 it could significantly lower, andtherefore this should be taken into account in the discussion below.

The APACHE II scoring system has been shown to be an accuratemeasurement of patient severity and correlates strongly with outcome incritically ill patients. (Knaus, W. A., E. A. Draper, D. P. Wagner, andJ. E. Zimmerman. 1985. APACHE II: a severity of disease classificationsystem. Crit Care Med 13:818-829; Moon, B. H., S. K. Park, D. K. Jang,K. S. Jang, J. T. Kim, and Y. M. Han. 2015. Use of APACHE II and SAPS IIto predict mortality for hemorrhagic and ischemic stroke patients. JClin Neurosci 22:111-115) APACHE II is measured during the first 24 h ofICU admission; the maximum score is 71. Patients with a score of 25 havea 50% predicted mortality rate, and those with a score over 35 have apredicted mortality rate of 80%. APACHE II scores for subjects in thecurrent study are presented in Table 31. The average score at diagnosiswas 12.3 (range 8-21). The probability of survival for the 10 individualpatients based on their APACHE II scores was calculated. (Rowan, K. M.,J. H. Kerr, E. Major, K. McPherson, A. Short, and M. P. Vessey. 1993.Intensive Care Society's APACHE II study in Britain and Ireland—I:Variations in case mix of adult admissions to general intensive careunits and impact on outcome. BMJ 307:972-977). Overall probability ofmortality was 16.8% (range (9.9-38.9%), with no significant differencesbetween the subgroups of patients who received one or two doses, betweenthe five pneumonia patients and three patients with biliary infection,or the two women versus the eight men. Current state-of-the-art pre-,intra-, and post-ICU treatment protocols for emergency admissions withsepsis may have improved physiological values and correctedphysiological abnormalities, resulting in lower mortality compared tothe rates predicted by the APACHE II scores. However, it should be notedthat each of the 10 patients enrolled in our study was in real danger ofmortality based on their admission APACHE II scores. The predictedprobability was that at least one study subject would die was 85%.

In patients with sepsis, an elevated SOFA score at presentation alsoreflects an increased risk of mortality. In one study thatincluded >180,000 patients hospitalized with sepsis, a SOFA score of 2-6at presentation (Table 31) corresponded to a predicted mortality of8-16%. (Raith, E. P., A. A. Udy, M. Bailey, S. McGloughlin, C. MacIsaac,R. Bellomo, D. V. Pilcher, Australian, O. New Zealand Intensive CareSociety Centre for, and E. Resource. 2017. Prognostic accuracy of theSOFA score, SIRS criteria, and qSOFA score for in-hospital mortalityamong adults with suspected infection admitted to the intensive careunit. JAMA 317:290-300).

In addition, a total of 37 matched controls were identified for the 10patients in the study based on the criteria of sepsis, admission toICU/IMU, source of infection, SOFA score ±2, age ±7 years, and gender.Table 31 shows the characteristics of controls compared to studysubjects. A comparison of survival between the study group andhistorical controls is shown in FIGS. 52A and 52B. Among the 37 matchedhistorical control patients, 10 died (27%). There was no significantdifference in mortality rates between Allocetra-OTS patients andhistorical controls (p-value using log rank test=0.078). FIGS. 52A and52B shows the Kaplan-Meier survival curve for all patients, andpneumonia-only patients. Although a bigger sample is needed to drawdefinite statistical conclusions regarding survival, based on thepredicted mortality by the APACHE II score, predicted high probabilityof at least one death in these 10 patients (85%), the predictedmortality by SOFA score at presentation, and mortality inmatched-historical controls, the study population would have expected tohave a mortality rate between 10-27%. The results of 0% mortality showno safety concerns in regard to mortality, with a potential preliminaryefficacy. This, with the SAE and AE profile described above encouragesthe investigators with regard to the safety profile of theinvestigational product.

Organ dysfunction.

Organ dysfunction improvement was a preliminary efficacy parametertested in this study. No residual organ dysfunction was seen in any ofthe 10 study subjects at 28 days. CNS. All patients finished 28 days offollow-up with a GCS of 15/15. Kidneys. Apart from patient 07, who wason chronic dialysis at admission, 3/9 patients (33%) had new-onset renalinjury and all had completely recovered to baseline kidney function asmeasured by creatinine level at 28 days. Lungs. 5/10 (50%) of patientshad lung involvement. No patient required mechanical ventilation. Allpatients recovered from lung dysfunction, had normal oxygen saturation,and needed no oxygen supplement at discharge. Cardiovascular. Threepatients had mean arterial pressure <70 mmHg but none neededvasopressors. Cardiac evaluation was made using clinical evaluation, ECGin all patients, troponin if indicated, and echocardiogram as indicated(needed in one patient). All patients had normal sinus rhythm ortachycardia upon screening, with no evidence of ischemia during theirillness. One patient with known paroxysmal atrial fibrillation had atransient episode of atrial fibrillation. Troponin was measured in 4patients and was normal in. One patient (01) underwent transthoracicechocardiography during his admission for the investigation of chestpain and elevated troponin levels following an episode ofsupraventricular tachycardia and electrical shock (DC cardioversion)before screening and entry to the study. Follow-up echocardiography inthis patient was performed at day 28. The results were comparable to thefirst evaluation. Hematological. Significant thrombocytopenia occurredin 8/10 patients (80%) with complete recovery in all. Liver.Hyperbilirubinemia occurred in four patients (40%) and three patientshad a biliary tract infection and all four had a complete recovery.Elevated liver enzymes (AST ALT) >3 of normal range were seen in 5/10patients with a complete recovery in all.

In the absence of days on respirator and days on vasopressors, we usedSOFA score to evaluate organ dysfunction. The SOFA score was introducedto describe organ failure severity in patients with sepsis, including a4□point assessment of dysfunction in each of six organ systems. (Minne,L., A. Abu-Hanna, and E. de Jonge. 2008. Evaluation of SOFA-based modelsfor predicting mortality in the ICU: A systematic review. Crit Care12:R161; Vincent, J. L., and R. Moreno. 2010. Clinical review: scoringsystems in the critically ill. Crit Care 14:207; Vincent, J. L., R.Moreno, J. Takala, S. Willatts, A. De Mendonca, H. Bruining, C. K.Reinhart, P. M. Suter, and L. G. Thijs. 1996. The SOFA (Sepsis-relatedOrgan Failure Assessment) score to describe organ dysfunction/failure.On behalf of the Working Group on Sepsis-Related Problems of theEuropean Society of Intensive Care Medicine. Intensive Care Med22:707-710). In patients with sepsis, a SOFA score ≥2 at presentationreflects clinically relevant organ dysfunction and an increased risk ofadverse outcomes. (Raith, E. P., A. A. Udy, M. Bailey, S. McGloughlin,C. MacIsaac, R. Bellomo, D. V. Pilcher, Australian, O. New ZealandIntensive Care Society Centre for, and E. Resource. 2017. Prognosticaccuracy of the SOFA score, SIRS criteria, and qSOFA score forin-hospital mortality among adults with suspected infection admitted tothe intensive care unit. JAMA 317:290-300). Apart for acute organinjury, we measured changes in the SOFA score from just before IPadministration, maximal SOFA score, AUC of SOFA score above baseline andtime to SOFA score <2 and compared it to the matched-historicalcontrols.

Interestingly, and as shown in Table 31 and FIGS. 55A, 55B, and 55C,despite the similarity of SOFA scores at entry (average of 3.4 versus3.47), the enrollment SOFA score was the highest for the treatedpatients that did not further progress (0 points) following treatment,while it progressed with a mean of +3.57 (range 0-15, median 1)(p<<0.0001, t-test and Wilcoxon) in the historical control population,suggesting inhibition of organ dysfunction deterioration withAllocetra-OTS treatment. In historical-matched controls with pneumoniait even went higher to a mean of +4.42 (range 015, median 4) (p<0.0001,t-test, and 0.0166, Wilcoxon).

The change in mean SOFA score was further measured between day 1 beforetreatment with Allocetra-OTS and at days 5, 7, and 28, (average deltaSOFA score between day 1-5, day 1-7, and day 1-28, p<0.0001, t test forall, and 0.0035, 0.001, and 0.0019, Wilcoxon, respectively, FIG. 55A).Also measured was the average change in the area under the curve (AUC)between days 1 and 5 and between days 1 and 7 that were all foundsignificantly ameliorated (average AUC between day 1-5 and day 1-7, bothp<0.0001, t test and 0.0011, Wilcoxon, FIG. 55B). Similar results wereobserved in patients with pneumonia. These data suggested that thefavorable clinical outcomes are reflected by lack of SOFA scoreprogression as manifested by maximal SOFA score (FIG. 55C), delta SOFAscore, and the average change in AUC.

Time in ICU/IMU and Hospital.

Since organ dysfunction was significantly improved and no mortalityoccurred, we were next interested to verify that these promising resultswere expressed in the duration of ICU/IMU and hospital stay. Since allpatients in the study group survived, we analyzed duration of stay inthe hospital and in ICU/IMU for all patients in the study and for allpatients in the matched historical control group who survived (FIG.55D). The length-of-stay in the hospital for patients treated with theAllocetra-OTS was significantly shorter with 11.3 days on average (range4-28, median 9) and for patients with pneumonia, 11.2 days, range 6-22,median 10), compared to an average for matched-historical controls of17.3 days (3-28, median 17) for all patients (p<0.0488, t-test, p<0.042,Wilcoxon), and average 18.84 days (range 6-28, median 19) for patientswith pneumonia (p<0.0552, t-test, p<0.0484, Wilcoxon). The ICUlength-of-stay for all treated patients was significantly shorter withan average of 4 days (range 2-7, median 3.5) and for patients withpneumonia 3.4 days (range 2-6, median 2) compared to 11.11 (1-28, median8) (p<<0.0001, t-test, p<0.092, Wilcoxon) and average of 13.89, (range1-28, median 11) in patients with pneumonia (p<<0.0001, t-test,p<0.0233, Wilcoxon). In addition, time to discharge from the hospitaland/or ICU/IMU (FIGS. 53A and 53B) was compared. Length ofhospitalization was analyzed as a time to event variable, comparing timeto discharge from the general hospital and the ICU between treatmentgroups. In this analysis, events of mortality were referred to as nodischarge event and the length of follow-up was censored at time ofdeath. Time to discharge was significantly shorter for the treated group(log rank, p=0.00085) for hospital discharge and for ICU/IMU discharge(log rank p=0.00096).

Exploratory Endpoints.

Effect of Allocetra-OTS on cytokines/chemokines/growth factors andimmuno-modulating agents. Pro-inflammatory cytokines. Pro-inflammatorycytokines regulate early responses to bacterial infection and mediatethe early acute phase in sepsis. Cytokines like IL-1, IL-6, and TNF-αact as endogenous pyrogens, upregulate the synthesis of secondarymediators and other pro-inflammatory cytokines by both macrophages andmesenchymal cells such as fibroblasts, epithelial and endothelial cells,and stimulate the production of acute-phase proteins or attractinflammatory cells. (Chaudhry, H., J. Zhou, Y. Zhong, M. M. Ali, F.McGuire, P. S. Nagarkatti, and M. Nagarkatti. 2013. Role of cytokines asa double-edged sword in sepsis. In Vivo 27:669-684). Eightpro-inflammatory cytokines were tested, including IL-6, TNF-α, IL-1β,IL-12p70, IL-18, IL-23, IFN-γ and IL-13; five of those were detectable(IL-6, TNF-α, IL-1β, IL-18, IFN-γ) and are presented here (FIG. 54A).FIG. 54A presents resolution of the cytokine storm in sepsis followingAllocetra-OTS administration.

Overall, all patients had elevated TNF-α and IL-6 levels at initialscreening and most had elevated IL-1β and IL-18, while some had elevatedIFN-γ; however, a gradual decrease for levels of all of the cytokinesstudies was observed upon resolution of sepsis. These trends ofpro-inflammatory cytokines were characteristic to most of the patients.

Anti-Inflammatory Cytokines.

Interestingly, anti-inflammatory cytokines are also upregulated insepsis in parallel to pro-inflammatory cytokines, and were suggested tohave a late contribution to sepsis-related immunosuppression.(Chousterman, B. G., F. K. Swirski, and G. F. Weber. 2017. Cytokinestorm and sepsis disease pathogenesis. Semin Immunopathol 39:517-528)Four anti-inflammatory cytokines were tested, including IL-10, IL-1Ra,IL-27, and soluble TNFR-1. IL-27 was not detected in our patients, andthe other anti-inflammatory cytokines are presented in FIG. 54B, upperpanel. Overall, the trend of the anti-inflammatory cytokines resembledthat of the pro-inflammatory cytokines. Most patients had elevatedIL-10, IL-1Ra, and TNFR-1, which gradually decreased as sepsis resolved.

Hematopoietic Growth Factors (HGFs).

During sepsis, immune cells undergo profound phenotypic modifications intheir activation state, response to stimuli, and localization. Thesephenomena are finely regulated by various cytokines and HGFs. An HGF isa relatively stable, secreted, or membrane-bound glycoprotein thatcauses immune cells to mature and/or proliferate. They also haveprofound effects on cell functions and behaviors. HGFs are deeplyinvolved in sepsis pathophysiology both in the initial and the latephases. (Chousterman, B. G., and M. Arnaud. 2018. Is there a role forhematopoietic growth factors during sepsis? Front Immunol 9:1015) Fourgrowth factors were tested in the study, including G-CSF, VEGF, GM-CSF(FIG. 55B) and LIF (a growth factor-like cytokine), which wasundetected. Overall, the trend of the HGFs resembled that of the pro-and anti-inflammatory cytokines and chemokines. Most patients presentedwith elevated G-CSF, VEGF, and to a lesser extent, GM-CSF at initialscreening; for all, a gradual decrease upon resolution of sepsis wasobserved. These trends of HGFs were characteristic of most patients thatreceived a single dose of Allocetra-OTS.

Chemokines.

Chemokines play pivotal roles in regulating the migration andinfiltration of monocytes/macrophages and neutrophils to sites ofinflammation, and as such, they are usually involved in sepsis. (Aziz,M., A. Jacob, W. L. Yang, A. Matsuda, and P. Wang. 2013. Current trendsin inflammatory and immunomodulatory mediators in sepsis. J Leukoc Biol93:329-342) Six chemokines were tested and are presented here, includingMCP-1, IP-10, MIP-1α, IL-8, Gro-β, and RANTES (FIG. 54C). Overall, mostpatients were screened with elevated MCP-1, IP-10, MIP-1α and IL-8; forall, a gradual decrease upon resolution of sepsis was observed. Ofexception were Gro-0 and RANTES, which were detected in most patientsbelow the normal levels and increased upon resolution of sepsis. Thesetrends of chemokines were characteristic to most of the patients thatreceived a single dose of Allocetra-OTS.

Other Immuno-Modulating Agents.

Several miscellaneous immuno-modulating factors were also tested as partof the immune modulating effect of Allocetra-OTS on the resolution ofsepsis (FIG. 54D). These factors included TREM-1, osteopontin, NGAL,gherlin, leptin, and several endocrinological hormones. Overall, all thepatients presented with elevated TREM-1 and OPN and most had elevatedNGAL and leptin levels; for all, a gradual decrease upon resolution ofsepsis was observed. Of exception is ghrelin, which was below the normallevels in all the patients at presentation and increased upon resolutionof sepsis. These trends of immuno-modulating factors were characteristicof most of the patients that received a single dose of Allocetra-OTS.

Endocrinology Kinetics.

Five hormones were tested in all subjects: Cortisol, FT3, FT4, Glucagonand Insulin. Additional hormones (TSH, ACTH and GH) were tested in somesubjects. Sepsis is considered an acute stress response with a releaseof stress hormones including cortisol and glucagon. There were elevatedcortisol levels in 7/10 patients at presentation, with the average ofall the patients being above normal (752 nmol/L, normal range of 140-690nmol/L). Patients 003 and 012 (both with pneumonia) had the highestlevels of cortisol at screening. Following infusion of theAllocetra-OTS, cortisol levels were downregulated (including Patient003), reaching normal concentrations by Day 28 (FIG. 54D).Interestingly, serum glucagon had similar kinetics to cortisol, and 7/10patients had high glucagon at screening (average level of allpatients=150 μg/ml, range 32-285 μg/ml) compared to normal (average of73 μg/ml, range 30-115 μg/ml), with a rapid decline in the first 2-3days reaching low levels on day 28. Insulin levels were in the lownormal range upon presentation (opposite to glucagon) and the levelswere normalized upon resolution of sepsis. ACTH and GH were tested insome subjects and were within the normal range throughout the study,except for Patient 007 (chronic dialysis) who had elevated ACTH levelson day 28 (21.5 pMol/L versus normal levels up to 13.5). All patientswere screened for FT3 and FT4 levels and all had low normal or belownormal FT3 levels with gradual increase thereafter as sepsis resolvedbut normal FT4 and TSH levels at presentation and during the resolutionof sepsis (FIG. 54D). These changes in serum thyroid function associatedwith acute illness have been termed “euthyroid sick syndrome,” or “lowT3 syndrome”. FT3 production during the acute stress response in sepsisis inhibited by both cortisol and IL-6. We therefore tested whether therecovery of FT3 seen in the patients was linked to the downregulation ofIL-6 and/or cortisol. Indeed, a strong negative correlation was foundbetween FT3 and IL-6 (p-Spearman=−0.73) and an intermediate negativecorrelation was found between FT3 and cortisol and glucagon(p-Spearman=−0.54, −0.56, respectively). The euthyroid sick syndromeshould thus not be viewed as an isolated pathologic event but as part ofa coordinated systemic reaction to sepsis involving both the immune andendocrine systems.

Discussion

In the current study, apoptotic cells were given within 24 hours ofdiagnosis of sepsis and initiation of antibiotic in order to modify theimmune response and thereby prevent organ dysfunction and possiblyshort- and long-term mortality. All patients suffered mild-to-moderatesepsis, with at least two systems involved and with a real-risk ofmortality estimated between 10-27% based on APACHE II, SOFA scores uponadmission, and matched-historical controls. Administration of apoptoticcells was safe and possibly efficacious, with no mortality in any of the10 patients, and with rapid resolution of the sepsis as judged by returnof organs function to baseline and rapid release from ICU/IMU andhospital. Advantages of the SOFA score compared with other ICU riskscores include its simplicity and ease of use, allowing it to becalculated daily at bedside without complex algorithms to demonstrateclinical improvement or deterioration over time. Daily trends in theSOFA score can predict adverse outcomes, because an increase in SOFAscore over time reflects progressive organ failure and higher risk ofdeath (Raith, E. P., A. A. Udy, M. Bailey, S. McGloughlin, C. MacIsaac,R. Bellomo, D. V. Pilcher, Australian, O. New Zealand Intensive CareSociety Centre for, and E. Resource. 2017. Prognostic accuracy of theSOFA score, SIRS criteria, and qSOFA score for in-hospital mortalityamong adults with suspected infection admitted to the intensive careunit. JAMA 317:290-300; Vincent and Moreno, 2010). Therefore, inaddition to traditional organ failure measurement, we intended toevaluate the ability of the SOFA score to predict organ dysfunction.

Apoptotic cells have immunomodulatory functions via their interactionwith macrophages and dendritic cells and their administration wassuggested to be used as a potential therapeutic intervention. (Mevorach,D., T. Zuckerman, I. Reiner, A. Shimoni, S. Samuel, A. Nagler, J. M.Rowe, and R. Or. 2014. Single infusion of donor mononuclear earlyapoptotic cells as prophylaxis for graft-versus-host disease inmyeloablative HLA-matched allogeneic bone marrow transplantation: aphase I/IIa clinical trial. Biol Blood Marrow Transplant 20:58-65;Trahtemberg, U., and D. Mevorach. 2017. Apoptotic cells inducedsignaling for immune homeostasis in macrophages and dendritic cells.Front Immunol 8:1356) Allocetra-OTS preparation is unique and differsfrom general populations of early apoptotic cells wherein the apoptoticcells are irradiated after induction of apoptosis an the population mayinclude apoptotic cells from different donors; this emphasizes earlyapoptosis and little to no necrotic cells or non-apoptotic proliferativecells (i.e. apoptotic cells are Annexin V⁺ propidium iodide-) in orderto avoid any necrotic cell effect. Although not all the molecular eventsunderlying the potential immune-regulating function of apoptotic cellsare clear, changes in macrophages and dendritic cells towards ahomeostatic phenotype have been investigated by several authors andimplicated in apoptotic cell-mediated immune modulation (Reviewed by(Trahtemberg, U., and D. Mevorach. 2017. Apoptotic cells inducedsignaling for immune homeostasis in macrophages and dendritic cells.Front Immunol 8:1356) Local administration of apoptotic cells has beenused to attenuate both bleomycin- and lipopolysaccharide (LPS)-inducedlung inflammation, with reduced neutrophil recruitment into the lung,enhanced phagocytosis by alveolar macrophages, and reducedpro-inflammatory cytokine production. Infusion of apoptotic cells 24hours after initiation of sepsis has also been shown to protect againstlethality in a mouse model of sepsis, with reduced pro-inflammatorycytokine and neutrophil recruitment into organs. Part of the beneficialeffect in the sepsis model was mediated by the direct binding of LPS byapoptotic cells, which led to the recognition and clearance ofLPS-covered apoptotic cells by macrophages in an anti-inflammatorymanner. However, the dose-dependent therapeutic use of apoptotic cellsin murine sepsis was found to be associated with a reduction in thecytokine storm and effect on the metabolome.

The possibility of blocking activity of a single cytokine was initiallywelcomed with excitement for its potential in sepsis therapy, but theexcitement has gradually waned. For example, TNF inhibitors have beeneffective as therapeutic anti-inflammatory agents and several TNFinhibitors have been approved by the Food and Drug Administration in theUSA for the treatment of rheumatoid arthritis, psoriatic arthritis, andCrohn's disease. However, clinical trials of anti-TNF therapy in sepsishave not been successful. (Bernard, G. R., B. Francois, J. P. Mira, J.L. Vincent, R. P. Dellinger, J. A. Russell, S. P. Larosa, P. F. Laterre,M. M. Levy, W. Dankner, N. Schmitt, J. Lindemann, and X. Wittebole.2014. Evaluating the efficacy and safety of two doses of the polyclonalanti-tumor necrosis factor-alpha fragment antibody AZD9773 in adultpatients with severe sepsis and/or septic shock: randomized,double-blind, placebo-controlled phase IIb study*. Crit Care Med42:504-511; Reinhart, K., T. Menges, B. Gardlund, J. Harm Zwaveling, M.Smithes, J. L. Vincent, J. M. Tellado, A. Salgado-Remigio, R.Zimlichman, S. Withington, K. Tschaikowsky, R. Brase, P. Damas, H.Kupper, J. Kempeni, J. Eiselstein, and M. Kaul. 2001. Randomized,placebo-controlled trial of the anti-tumor necrosis factor antibodyfragment afelimomab in hyperinflammatory response during severe sepsis:The RAMSES Study. Crit Care Med 29:765-769) In addition to anti-TNFtherapy, several research efforts have been dedicated to investigatingthe potential of therapies targeting pro-inflammatory cytokines insepsis. Blockade of IL-17, (Takahashi, N., I. Vanlaere, R. de Rycke, A.Cauwels, L. A. Joosten, E. Lubberts, W. B. van den Berg, and C. Libert.2008. IL-17 produced by Paneth cells drives TNF-induced shock. J Exp Med205:1755-1761) IL-1, (Wakabayashi, G., J. A. Gelfand, J. F. Burke, R. C.Thompson, and C. A. Dinarello. 1991. A specific receptor antagonist forinterleukin 1 prevents Escherichia coli-induced shock in rabbits. FASEBJ 5:338-343) and other cytokines was protective in experimental modelsof sepsis.

However, more than 40 clinical trials on anti-cytokine therapeuticagents have failed, implying a significant disconnect betweenexperimental and clinical sepsis. (Cohen, J., S. Opal, and T. Calandra.2012. Sepsis studies need new direction. Lancet Infect Dis 12:503-505)This emphasizes the uniqueness of the current data presented hereinshowing treatment of clinical sepsis using Allocetra-OTS.

The mechanism of action of apoptotic cells is based on their known andwell-studied interaction with monocytes/macrophages and dendritic cells,with general downregulation of their exaggerated activity. There is awide-spectrum effect on cytokines/chemokines but not absolute blocking,as seen in anti-cytokine therapy. (Trahtemberg, U., and D. Mevorach.2017. Apoptotic cells induced signaling for immune homeostasis inmacrophages and dendritic cells. Front Immunol 8:1356) Indeed, TNF,IL-1β, and IL-6 are recognized as the main cytokines that mediate theinitial pro-inflammatory response of the innate immune system to injuryor infection. Both TNF and IL-1β activate endothelial cells, attractingcirculating polymorphonuclear leukocytes (PMNs) to the site. They alsoenter the circulation, causing fever and other systemic symptoms. IL-6enhances the liver's production of acute phase reactants, including CRP,and stimulates a shift in the production of cells in the bone marrow sothat more PMNs are produced. Therefore, these three cytokines areessentially responsible for the features of what was called the systemicinflammatory response syndrome (SIRS) and could be potentially useful asbiomarkers of sepsis. (Faix, J. D. 2013. Biomarkers of sepsis. Crit RevClin Lab Sci 50:23-36) Indeed, all three were elevated in variabledegrees, according to the nature of infection, personal immune response,and timing of emergency room arrival, in the patients in this study.

Interestingly, and as suggested elsewhere, (Angus, D. C., and T. van derPoll. 2013. Severe sepsis and septic shock. N Engl J Med 369:840-851)anti-inflammatory cytokines are also elevated early in the course ofsepsis. IL-10 is a major anti-inflammatory cytokine that is produced bymany types of immune cells, such as monocytes, macrophages, B- andT-lymphocytes, and NK cells. IL-10 suppresses the production ofpro-inflammatory mediators, such as TNF-α, IL-1, IL-6, IFN-γ, and GM-CSFin immune cells. (Schulte, W., J. Bernhagen, and R. Bucala. 2013.Cytokines in sepsis: potent immunoregulators and potential therapeutictargets—an updated view. Mediators Inflamm 2013:165974) High IL-10levels are also associated with more important features ofsepsis-induced immunosuppression. (Chousterman, B. G., F. K. Swirski,and G. F. Weber. 2017. Cytokine storm and sepsis disease pathogenesis.Semin Immunopathol 39:517-528) Patients with septic shock showedincreased levels of IL-10 compared to healthy controls, and L-10 wasalso positively correlated with the pro-inflammatory mediators IL-6,IL-8, MCP-1, MIP-10, IFN-γ, and GM-CSF. This supported the notion thatsecretion of pro- and anti-inflammatory mediators in septic shock occursas a simultaneous immune response program initiated early in the courseof the disease. (Tamayo, E., A. Fernandez, R. Almansa, E. Carrasco, M.Heredia, C. Lajo, L. Goncalves, J. I. Gomez-Herreras, R. O. de Lejarazu,and J. F. Bermejo-Martin. 2011. Pro- and anti-inflammatory responses areregulated simultaneously from the first moments of septic shock. EurCytokine Netw 22:82-87) In severe sepsis, the IL-10/lymphocyte ratio wassignificantly correlated with the APACHE II score and strongly predicted28-day mortality. (Li, X., Z. Xu, X. Pang, Y. Huang, B. Yang, Y. Yang,K. Chen, X. Liu, P. Mao, and Y. Li. 2017. Interleukin-10/lymphocyteratio predicts mortality in severe septic patients. PLoS One12:e0179050)

The three measured anti-inflammatory cytokines that are associated withsevere sepsis and decreased survival were shown to be elevated uponscreening, in parallel to the pro-inflammatory cytokines. They wereclearly downregulated following the IP administration with gradualkinetics towards normal levels and were associated with resolution ofthe septic condition and rapid release from ICU/IMU and the hospital.Importantly, although apoptotic cells may cause an increase inanti-inflammatory cytokines, there was no increase in the measuredanti-inflammatory cytokines in our patients. Instead, they did notinhibit and possibly facilitated resolution of the abnormalanti-inflammatory response. This property of apoptotic cells, ofrebalancing the immune system and pro-homeostatic effect, was clearlyseen in the resolution of inflammation in this study. Most elevatedcytokines went back to normal levels, without reaching below normal, ascan be seen when treating with an antibody against a specific cytokine.

IL-8 is a key chemokine, secreted by multiple cell types, includingmonocytes, neutrophils, epithelial cells, fibroblasts, endothelialcells, mesothelial cells, and tumor cells. Its release is stronglyinduced by IL-1 and TNF-α. IL-8 plays an important role in inflammationand wound healing, and has the capacity to recruit T cells as well asrecruiting and activating neutrophils. (Qazi, B. S., K. Tang, and A.Qazi. 2011. Recent advances in underlying pathologies provide insightinto interleukin-8 expression-mediated inflammation and angiogenesis.Int J Inflam 2011:908468) In the current study, most patients hadelevated classical chemokines in addition to the expected low levels ofRANTES at initial screening, in parallel with elevated levels of pro-and anti-inflammatory cytokines. Factors with abnormally high levelswere clearly downregulated and RANTES was upregulated following IPadministration with gradual kinetics towards normal levels. Thesechanges were associated with resolution of the septic condition andrapid release from ICU/IMU and the hospital.

G-CSF levels are low in steady-state and rise after inflammatorystimuli. High levels of G-CSF are induced by TNFα, IL-1, or LPSstimulation of macrophages or epithelial cells. T cells can also induceG-CSF production through IL-17 release. The main effects of G-CSF are toinduce proliferation and differentiation, but also the survival of cellsin the neutrophil lineage. G-CSF also enhances neutrophil production ofcytokines, production of ROS, and phagocytosis when added to otherstimulation, and it enhances mobilization of neutrophils in a direct andindirect chemotactic effect. (Chousterman, B. G., and M. Arnaud. 2018.Is there a role for hematopoietic growth factors during sepsis? FrontImmunol 9:1015) Elevated G-CSF levels were found in sepsis and trauma.Interestingly, and not in the patients treated with Allocetra-OTS inthis study, increased levels remained high for a longer duration insepsis. (Tanaka, H., K. Ishikawa, M. Nishino, T. Shimazu, and T.Yoshioka. 1996. Changes in granulocyte colony-stimulating factorconcentration in patients with trauma and sepsis. J Trauma 40:718-725;discussion 725-716)

Summary:

Ten patients were treated with Allocetra-OTS, administered as a singledose or two sequential doses. All 10 patients had mild to moderatesepsis with a SOFA score range of 2-6 upon entering the study. Noserious adverse events (SAEs) and no related AEs were reported. All 10study subjects survived while matched historical controls had amortality rate of 27%. The study subjects exhibited rapid resolution oforgan dysfunction and had significantly shorter ICU lengths of staycompared to matched historical controls (p<0.0001). All patients hadboth elevated pro- and anti-inflammatory cytokines, chemokines andadditional immune modulators that gradually decreased followingtreatment.

Brieftly, at initial screening, most patients had clearly elevatedlevels of most pro- and anti-inflammatory cytokines, chemokines (apartfrom RANTES and Gro-b), and HGFs, which were downregulated following IPadministration and this correlated with the resolution of sepsis. Thesefindings agree with the clinical resolution of sepsis in these 10patients and the expected effect of Allocetra-OTS onmonocytes/macrophages and dendritic cells. (Trahtemberg, U., and D.Mevorach. 2017. Apoptotic cells induced signaling for immune homeostasisin macrophages and dendritic cells. Front Immunol 8:1356)

Each of these pro-inflammatory cytokines was proven to be pathogenic insepsis and septic shock; however, single targeting of these cytokinesdid not ameliorate sepsis in many trials using anti-TNF, anti-IL-1b, andother cytokines in sepsis. (Cohen, J., S. Opal, and T. Calandra. 2012.Sepsis studies need new direction. Lancet Infect Dis 12:503-505) In thatregard Allocetra-OTS represents a more holistic approach, leading torebalancing of all pro- and anti-inflammatory cytokines and chemokines,growth factors, and other immuno-modulating agents, and reprogramming ofmonocytes/macrophages and dendritic cells. (Trahtemberg, U., and D.Mevorach. 2017. ibid) In order to further evaluate their effect, arandomized-controlled trial is needed, but these results may reflect anovel and safe mechanism for treatment of sepsis as well as flucomplications that are characterized by cytokine storm, and cytokinerelease syndrome that characterized chimeric antigen receptor (CAR)-Tcell therapy.

Conclusion.

Administration of Allocetra-OTS to patients with mild-to-moderate sepsiswas safe and had a significant immuno-modulating effect, leading toearly resolution of the cytokine storm. There were indications that thistreatment may also be efficacious based on comparisons with mortalityscore prediction and historical matched-controls, and the resolution oforgan dysfunction outcomes compared to matched historical controls.

While certain features disclosed herein have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritdisclosed herein.

What is claimed is:
 1. A method of treating, preventing, inhibiting,reducing the incidence of, ameliorating, or alleviating, sepsis-relatedorgan dysfunction, or any combination thereof, in a subject in need,comprising the step of administering a composition comprising an earlyapoptotic cell population to said subject, wherein said early apoptoticcells comprise peripheral blood mononuclear cells, wherein at least twoorgan systems are dysfunctional in said subject, and wherein saidadministering treats, prevents, inhibits, reduces the incidence of,ameliorates, or alleviates sepsis-related organ dysfunction in saidsubject.
 2. The method of claim 1, wherein sepsis comprises mild,severe, acute, or highly aggressive sepsis.
 3. The method of claim 1,wherein the survival of said subject is increased.
 4. The method ofclaim 1, wherein said method reduces the incidence of organ dysfunction.5. The method of claim 1, wherein organ dysfunction comprises multipleorgan dysfunction or acute multiple organ dysfunction syndrome (MODS).6. The method of claim 1, wherein said early apoptotic cell populationcomprises a mononuclear apoptotic cell population comprising a decreasedof non-quiescent non-apoptotic cells, a suppressed cellular activationof any living non-apoptotic cells, or a reduced proliferation of anyliving non-apoptotic cells, or any combination thereof.
 7. The method ofclaim 1, wherein said early apoptotic cell population comprises a pooledpopulation of early apoptotic cells.
 8. The method of claim 1, whereinsaid administration of the early apoptotic cell population is within 24hours of initiation of sepsis.
 9. The method of claim 1, wherein saidadministering comprises a single infusion of said early apoptotic cellpopulation or multiple infusions of said early apoptotic cellpopulation, said composition comprising 40-240×10⁶ cells/kg earlyapoptotic cells.
 10. The method of claim 1, wherein said administeringcomprises intra venal administration.
 11. The method of claim 1, furthercomprising administering an additional therapy.
 12. The method of claim11, wherein said additional therapy is administered prior to, concurrentwith, or following administration of said early apoptotic cells.
 13. Themethod of claim 1, wherein said method comprises a first-line therapy oran adjuvant therapy.
 14. The method of claim 1, wherein said methodcomprises rebalancing the immune response of said subject.
 15. Themethod of claim 14, wherein said rebalancing comprises reducing thesecretion of one or more proinflammatory cytokine/chemokine, or reducingthe secretion of one or more anti-inflammatory cytokines/chemokines, ora combination thereof.
 16. The method of claim 1, wherein said methodprevents, inhibits, reduces the incidence of, or reduces the severity ofa cytokine and chemokine storm in said subject.
 17. The method of claim1, wherein said at least two organ systems are selected from kidney,lung, cardiovascular, and liver.
 18. The method of claim 9, wherein saidcomposition comprises 140×10⁶ ±20% cells/kg early apoptotic cells. 19.The method of claim 11, wherein said additional therapy comprisesadministering an antibiotic.
 20. The method of claim 19, wherein saidantibiotic is ertapenem.